Therapeutic suppression of specific immune response by administration of oligomeric forms of antigen of controlled chemistry

ABSTRACT

A method of making a non-immunogenic construct for reducing a non-primary antibody response to an epitope of a T-dependent antigen by coupling two or more copies of the epitope to a nonimmunogenic soluble carrier to yield a conjugate preparation and removing high molecular weight immunostimulatory molecules.

This application is a continuation of application Ser. No. 08/391,267,filed Feb. 21, 1995 and now U.S. Pat. No. 6,022,544, which is acontinuation of application Ser. No. 07/808,797 filed Dec. 17, 1991 andnow abandoned, which is a continuation-in-part of application Ser. No.07/628,858, filed Dec. 17, 1990 and now abandoned, which is acontinuation-in-part of application Ser. No. 07/354,710, filed May 22,1989 and now abandoned, which is a continuation in part of applicationSer. No. 07/248,293, filed Sep. 21, 1988 and now U.S. Pat. No.5,126,131, which is a continuation of application Ser. No. 06/869,808,filed May 29, 1986 and now abandoned, which is a continuation ofapplication Ser. No. 06/460,266, filed Jan. 24, 1983 and now abandoned.

The present invention relates, in general, to a method of suppressing anundesired immune response and to constructs suitable for use therein.

The invention described herein was made in part in the course of workunder a grant or award from the United States Army, No. DAMD17-86-C-6038.

As a mechanism of self defense, animals have developed a complex set ofresponses to foreign material, collectively called the immune system.Immune responses are generally advantageous (protective) in nature,however, under certain situations, the animal body produces an immuneresponse that is undesirable. Examples of such undesirable responsesinclude allergic reactions, characterized by the production of IgEantibodies to extrinsic antigens, and autoimmune diseases in which theimmune system reacts against self antigens.

During the past few decades, a number of methods have been described forinhibiting, suppressing or “curing” specific immune responses. Thesemethods involve the treatment of animals with different kinds ofchemical preparations, the details of which are described below. Theimmune modification methodology which forms the basis of the presentseries of applications is based on the premise that the immune systemrecognizes foreign antigens in the context of physically constrainedarrays. In order to stimulate the immune system, arrays must exceed aspecific size (or geometry) and have a minimum number of physicallyaccessible epitopes which are identical in nature (minimum valence).Once these two parameters are met or exceeded, the immune system willrespond by the production of antibodies (IgM, IgG and/or IgE) by antigenspecific B-cells and by the production of T-cell factors and/oractivities (T-cell ‘help’, cytokines, cytoxicity, etc.).

The method to which the present invention relates is based on thefinding by Applicants that this system can be manipulated by introducingsynthetically derived macromolecular arrays that are “subthreshold” ingeometry and/or valence and that are designed to compete with naturallyoccurring arrays for the suppression of autoimmune and extrinsicallergic responses.

The technology which forms the basis of the invention is derived fromthe Immunon model of immune response described by Dintzis et al in Proc.Nat'l. Acad. Sci. USA, 73:3671-3675 (1976). That paper discloses theconcept of there being a threshold as to the number and spacing ofhaptens on T-cell independent antigens in order to obtain an immunogenicresponse. The 1976 paper also discloses that the non-immunogenicpolymers are suppressive of the action of immunogenic polymers towardstriggering the de novo immune response in non-immunized animals. Thesuppressive effect of non-immunogenic polymers on the immunogenicresponse of immunogenic polymers is further described in Proc. Nat'l.Acad. Sci. USA, 79:395, 1982; Proc. Nat'l. Acad. Sci. USA, 79:884, 1982;and J. Immunol., 131:2196, 1983. (See also Dintzis et al, J. Immunol.135:423, 1985; Dintzis et al In: Theoretical Immunology, Pt. 1, Vol. II.ed. Perelson, A. S. Addison-Wesley Publishing Co., Reading, Mass. pp83-103, 1988; Dintzis et al, J. Immunol. 143:1239, 1989; Dintzis et al,Eur. J. Immunol. 70:229, 1990; and Dintzis and Dintzis, Immunol. ReviewsNo. 115, pp 243-251, 1990).

The earlier applications of the present series include details ofstudies that were done using experimental paradigms involvingT-independent antibody responses which can be assessed by the level ofIgM production. The use of size restricted backbones of various types(linear polyacrylamide, dextran, Ficoll, carboxymethyl cellulose, etc.)to suppress IgM antibody production to small molecular weight haptenssuch as DNP and fluorescein is specifically described. (See Examples 1to 7 below.) In addition, reference is made in the earlier filings tothe use of the present invention to suppress allergies to pollen andauto-immune disease, including multiple sclerosis and myasthemia gravis.The present application includes details of studies relating to T-celldependent antibody production as well as T-cell responses by themselves.The data presented herein thus further support the applicability of theimmune suppression methodology of the earlier filed applications in thisseries to complex responses involving T-cell dependent antibodyproduction, represented by IgG and IgE. In addition, the presentdisclosure underscores the desirability of characterizing thesuppressive constructs to ensure that they are free from potentiallysimulatory molecules.

As indicated above, varying chemical preparations reportedly suitablefor use in methods of inhibiting immune responses have been the subjectof numerous publications. The methods disclosed are apparently based onthe “special chemical composition” of the polymeric backbone materialused which forms an epitope carrier. The mechanisms by which theobserved specific immune suppresion occurs, and the specific molecularattributes inferred to bring about the suppression, have been variouslyascribed to:

1) chemical composition as determined by the ratios of carbon tohydrogen to oxygen in the carrier material (Dawn et al, J. Immunol.126:407-413, (1981); Wei et al, Int. Archs. Allergy Appl. Immunol.85:1-7 (1988)).

2) “unnaturalness” as defined by the use of the “unnatural” D-aminoacids, rather than “natural” L-amino acids in synthesizing thepolypeptide carrier substance (Katz et al, J. Exp. Med., 134:201-223(1971); Liu et al, Proc. Natl. Acad. Sci. USA 76:1430-1434 (1979); Liuet al, J. Allergy Clin. Immunol. 66:322-326 (1980));

3) “special” chemical properties, undefined in nature; and

4) ability to increase “specific suppressor cells” in undefined ways.

(See specific comments that follow). To the best of Applicants'knowledge, however, no other group has proposed that immune suppressionoccurs because the suppressive material contains molecules with theproper combination of molecular size and epitope valence and, thus, noother group has taught or even suggested the method to which the presentinvention relates.

Sehon and coworkers have carried out a number of studies of specificimmune suppression, induced by the injection of polymeric moleculescomposed of epitopes coupled to a polyvinylalcohol (PVA) backbonestructure (see, for example, Dawn et al, J. Immunol. 126:407-413 (1981);Wei et al, Int. Archs. Allergy Appl. Immunol. 85:1-7 (1988)). The PVAbackbone structure was created by reacting low molecular weight PVA, 14kDa, with cyanogen bromide to convert some of the hydroxyl groups on thepolymer to a reactive form, and coupling those activated hydroxyl groupsto amino groups on aliphatic diamine. This reaction was expected by theauthors to substitute the PVA polymer molecules with a number of freealiphatic amino groups from the unreacted ends of the diamine adduct.These ends were subsequently substituted with hapten groups to formmultiply substituted PVA molecules of molecular weight supposedly almostunchanged from that of the original PVA.

This empirical procedure produced soluble haptenated polymeric materialwhich was suppressive of specific immune responses against the hapteninvolved. However, in reacting a multiply reactive polymer (cyanogenbromide activated PVA) with an excess of a divalent reactant(ethylenediamine) a very substantial amount of cross-linkage between thepolymer molecules occurred with the resulting formation of multiplycross-linked molecules of a wide range of molecular weights. AlthoughSehon and Lee noted that precipitates formed, and discarded them, theyapparently did not take this as an indication that higher molecularweight (and thus potentially stimulatory) polymers were being produced.

Applicants have, in fact, reported, (Dintzis et al, J. Immunol.143:1239-1244 (1989)) that higher molecular weight (over 100 kDa) PVAmolecules multiply substituted with hapten are immunogenic in vivo andin vitro, giving bell shaped dose response curves. Similar moleculeswith molecular weights below 100 kDa, however, were found by Applicantsto be inhibitory of the immune response, without having stimulatorycapacity, as predicted by their paradigm.

Katz and co-workers have described the specific suppression of theimmune response to epitopes by treatment with polymer preparationscomposed of those epitopes coupled to a carrier backbone made of thesynthetic polypeptide, poly(D-glutamic acid, D-lysine) or poly(D-Glu,D-Lys) (see, for example, (Katz et al, J. Exp. Med. 134:201-223 (1971);Liu et al, Proc. Natl. Acad. Sci. USA 76:1430-1434 (1979); Liu et al, J.Allergy Clin. Immunol. 66:322-326 (1980)). This polypeptide is acommercially available randomly ordered polymer synthesized fromchemically activated forms of the D-amino acids, D-lysine and D-glutamicacid, in the ratio 60:40. Katz has rationalized the findings of immunesuppression as caused by the “unnatural” character of the syntheticpolypeptide composed of the unusual D-amino acids rather than the usualL- forms of the amino acids, which are found in all protein molecules.This interpretation was apparently supported by the finding thatequivalent immune suppression was not observed when the carrier backbonepolypeptide was synthesized from the more normal L-amino acids.

The findings of Katz fit well into the Immunon paradigm as illustratedbelow:

1) The poly(D-Glu,D-Lys) preparation used by Katz as a backbone polymerwas obtained from commercial sources, and had average molecular weightof less than 100 kDa (the primary commercial producers, Yeda (in Israel)and Sigma (in St. Louis), have informed Applicants that it is notpossible for them to produce such polymers with average molecularweights greater than 70 kDa). Thus, Katz apparently used polymers ofmolecular weight less than 100 kDa as suppressive backbone material,without realizing the significance of this fortitous choice of molecularweight.

2) Starting with the highest molecular weight poly(D-Glu,D-Lys)available, approximately 70 kDa from Yeda, Applicants substituted anumber of the lysine amino groups with the hapten, fluorescein and foundthe resulting FLU-poly(D-Glu,D-Lys) to be non-immunogenic, as expected.Examination of the material by HPLC revealed that, as expected, itcontained molecules with a wide range of molecular weights, from under40 kDa to a small amount over 100 kDa. When size fractionated by gelfiltration chromatography on Superose CL-6B columns, it was possible toseparate out a small amount of material of molecular weightapproximately 200 kDa. This higher molecular weight fraction proved tobe immunogenic for an immune response against fluorescein in mice. Thisfinding indicates that there is nothing intrinsically suppressive aboutFLU-poly(D-Glu,D-Lys), but that it can be stimulatory ornon-stimulatory, depending on the molecular size.

3) To further test the effect of molecular size, Applicants cross-linkednon-immunogenic 70 kDa FLU-poly(D-Glu,D-Lys) molecules withcarbodiimide, coupling some carboxyl groups on glutamic acid residueswith amino groups on lysine residues to form stable amide bonds. A widerange of molecular weight products resulted. When these weresize-fractionated on gel filtration columns, the material with molecularweights well above 100 kDa were immunogenic both in vivo and in vitro,whereas the fractions with molecular weights under 100 kDa was notimmunogenic. This again fits the expectations of the Immunon hypothesis,and is not consistent with the interpretations put forth by Katz.

4) Since the mammalian body does not produce enzymes capable ofhydrolyzing polypeptides composed solely of D-amino acids, it is to beexpected that such polypeptides, whether free or epitope substituted,will not be rapidly degraded in the animal body, and will be longlasting in their effects. However, polypeptides made of the usualL-amino acids can be rapidly hydrolyzed by normal proteolytic enzymesand would not be expected to have sustained effects. This suggests thatthe properties ascribed by Katz to the “unnatural” nature of the D-aminoacid polypeptide are due solely to the resistance to enzymaticbreakdown, a characteristic shared by many synthetic and naturalpolymeric molecules.

Diener and co-workers have published a number of papers describing thespecific suppressive immune effects of epitopes coupled tocarboxy-methylcellulose as carrier (see, for example, Diner et al, J.Immunol. 122:1886-1891 (1979)). These have been ascribed by Diener tothe special chemical nature of carboxymethyl cellulose, withoutconsideration of the molecular weight of the material. However,Applicants have reported that haptenated preparations of carboxymethylcellulose of molecular weights under 100 kDa are suppressive forepitopes coupled to them, without being stimulatory at any dose, whereaspreparations of molecular weights over 100 kDa are stimulatory at properdoses (Dintzis et al, J. Immunol. 143:1239-1244 (1989)). Apparently,Diener used material of molecular weight predominately under 100 kDa,without realizing the significance that the molecular size of thepolymers had on the immune effects of his preparations.

The specific suppressive effect of hapten coupled topolyvinylpyrrolidone (PVP), a material which has been used as a bloodsubstitute has been reported (von Specht et al, Clin. Exp. Immunol.33:292-297 (1978); Lee et al, Eur. J. Immunol. 11:13-17 (1981)). Otherauthors have published on similar suppressive effects of haptens coupledto Ficoll (Watanabe et al, J. Immunol. 118:251-255 (1977)), pneumococcalpolysaccharides (Borel et al, Nature 261:49-50 (1976); Mitchell et al,Eur. J. Immunol. 2:460-467 (1972)), plant polysaccharides (Moreno et al,Clin. Exp. Immunol. 31:499-511 (1978)); Humphrey, Eur. J. Immunol.11:212-220 (1981)) or isologous immunoglobulin (Lee et al, J. Immunol.114:829-842 (1975); Borel et al, Nature 261:49-50 (1976)). These reportsare quite diverse, but do not address the combined effects of themolecular weight of polymer carrier and the epitope valence on theimmune response which results from their administration, as Applicantshave done. Molecular weight characterization of the epitope-substitutedpolymer preparations was not done in these published studies. However,the experimental protocols are consistent with the interpretation thatthe average molecular weights of these preparations was under 100 kDa inall instances.

In general, authors who have reported specific suppressive effects fromhapten-coupled polymer preparations have apparently chanced uponpreparations which fit Applicants' description of suppressive solublemolecules, namely a substantial number of epitopes coupled to a solublepolymeric carrier of molecular weight less than about 100 kDa. Whilethese conditions may be unwittingly encountered under a variety ofcircumstances, as noted above, such encounters are not suggestive of thepresent invention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Conversion of dextran to GMB-dextran.

FIG. 2. Conversion of carboxymethyl dextran to dexamine.

FIG. 3. Conversion of dextran to polyanionic dexamine.

FIG. 4. Conversion of dexamine to polycationic dexamine.

FIG. 5. Conversion of poly(acrylamide, acrylic acid) to primary aminecontaining poly(acrylamide, acrylic acid).

FIG. 6. Examples of trimeric and tetrameric “point source” scaffolds.

FIG. 7. Cyclodextrins as point source scaffolds.

FIG. 8. Conjugation of peptides to GMB-dextran.

FIG. 9. Analytical equilibrium ultracentrifugation of fluoresceinateddextrans.

FIG. 10. Amino acid analysis of a peptide-dextran conjugate.

FIG. 11. Lupus histone peptide-dextran conjugate analysis.

FIG. 12. Conjugate peptide substitution density equation.

FIG. 13. Alternative marker amino acids derived from acid hydrolysis ofpeptide-dextran conjugates.

FIG. 14. Size-fractionated DNA/Dex_(7 0K) conjugate hydrolysis peaksproduced as a result of acid (HCL) hydrolysis and subsequentderivatization with phenylisothiocyanate (PITC).

FIG. 15. DNA hydrolysis peaks produced as a result of acid hydrolysisand subsequent derivatization with phenylisothiocyanate (PITC).

FIG. 16. Dose-response measurements showing the mean of the relativeconcentration, in serum from individual mice, of IgM antibody againstDnp at 6 days after injection of a stimulatory polymer (polymer S) inamounts shown (10 BALB/c mice per point). Error bars indicate SEM whenit is larger than the circle. The solid curve gives the theoreticalresponse expected from Eq. 1 (see page 96) for a peak response occurringat a dose of 0.3 μg per mouse and an immunon size, q, of 10. Thetheoretical response is not sensitive to the value of q if q is greaterthan five. The peak of the response curve corresponds to approximately30 μg of anti-Dnp IgM per ml of serum.

FIG. 17. Dose-response measurements for different lots of BALB/c mice.Measurements were made on serum from individual mice. The mean ofmeasurements on each group at each dose is shown, together with the SEMwhen it is larger than the symbol. Of the symbols used, the solid blackdot represents ten mice per point (these points being the same as inFIG. 16); the open circle “∘” represents five mice per point; and thesymbol ▪ represents six mice per point.

FIG. 18. Response-reduction measurements for increasing doses ofnonimmunogenic polymer preparation N injected simultaneously with aconstant dose of immunogenic polymer preparation S. Measurements weremade on serum from individual mice. The mean of each group is showntogether with the SEM when it is larger than the symbol. BALB/c mice, 10mice per point; 0.31 μg of polymer S given to each mouse. The solidcurve gives the theoretical response expected from Eq. 1 for an immunonsize, q, of 10 and D_(S) ^(max) set equal to 0.5 μg per mouse as derivedfrom FIG. 17. The theoretical response is quite insensitive to the valueof q but is shifted left or right according to the value of D_(S)^(max), with no change in shape.

FIG. 19. Dose-response measurements regarding the relative number ofdirect anti-Dnp plaques produced from spleen cell cultures 3 days afterthe start of incubation in the presence of various concentrations ofimmunogenic polymer S. The data represent the mean of duplicate cultureswith triplicate assays per culture; and SD is indicated when it islarger than the circle. The experimental peak response corresponds to≈300 plaques per 10⁶ spleen cells with a blank (without polymer) of ≈20plaques per 10⁶ spleen cells. The curve gives the theoretical responseexpected from Eq. 1 for a peak response occurring at a polymerconcentration of 0.4 ng/ml and an immunon size, q, of 10.

FIG. 20. Dose-reduction measurements for increasing doses ofnonimmunogenic polymer preparation N incubated in spleen cell culturewith a constant dose (0.3 ng/ml) of immunogenic polymer preparation S.Procedures and data treatment were as in FIG. 19. The different symbolsshow data obtained in separate experiments. The solid curve gives thetheoretical response expected from Eq. 1 for an immunon size, q, of 10and D_(S) ^(max) set equal to 0.4 ng/ml as derived from FIG. 19.

FIG. 21. In vitro response kinetics. The direct (IgM) anti-Fl responseof naive spleen cells to increasing doses of Fl₁₁₀PVA400 was measuredafter 3,4, or 5 days of culture. All the S.D. were less than 10%.

FIG. 22. in vivo response kinetics. An optimal dose of Fl₅₅PVA200 (10ug/mouse) was injected i.p. in 0.5 ml saline, and direct (IgM) anti-Flresponse was measured at times from 0 to 66 days. Each point represents3 mice and is the mean of triplicate assays. The S.D. was less than 10%.

FIG. 23. In vivo normalized dose-response curves generated by fourFl-polymers with different carriers. Each point represents the mean oftriplicate assays. Mice were injected with increasing doses of aFl-polymer i.p. in saline (three mice/point), and PFC were measuredafter 4 days. Curves were normalized so that maximum response wasassigned a value of 1, and other responses were expressed as fractionsof the maximum response. The S.D. was less than 10%.

FIG. 24. In vitro normalized dose-response curves generated by fiveFl-polymers with different carriers. Direct (IgM) anti-Fl PFC weremeasured after three days of culture of polymer with naive spleen cells.Curves were normalized so that maximum response was assigned a value of1, and other responses were expressed as fractions of the maximumresponse.

FIG. 25. Inhibition of the in vitro response to Fl₉₀Fic750 bynon-stimulatory Fl-polymers. The IgM PFC response to Fl₉₀Fic750 alonewas assigned a value of 1 and the response of cultures containing addedamounts of nonimmunogenic polymers was expressed as the fractionalrelative response. The concentration of Fl₉₀Fic750 in each culture waskept constant at 3 ng per ml.

FIG. 26. Inhibition of the in vitro response to Fl₉₀Fic750 by high dosesof immunogenic Fl-polymers. The IgM PFC response to 3 ng per ml ofFl₉₀Fic750 alone was assigned a value of 1, and the response of culturescontaining added amounts of Fl-polymer was expressed as the fractionalrelative response.

FIG. 27. Anti-fluorescein IgG antibody serum levels in immunized mice asa function of time. The mice have been repeatedly injected withfluoresceinated ovalbumin on aluminum hydroxide adjuvant and allowed torest for several weeks before the bleedings shown on the Figure. Allbleedings were analyzed in the same ELISA assay at serum dilutions of10,000 fold.

FIG. 28. Cure of anti-fluorescein IgG serum antibody level by thefluoresceinated polyacrylamide polymer FL30-Pa50, i.e., a 50 kDpolyacrylamide polymer substituted with 30 fluorescein hapten groups.The time at which the dose of 3 mg of polymer was given has beenarbitrarily designated day 0 on the time scale. The open circle datapoints are the averages of the data points shown in FIG. 27 forunsuppressed mice, with standard deviations and a least square fitstraight line indicated.

FIG. 29. Cure of anti-fluorescein IgG antibody level by thefluoresceinated dextran polymer FL25-Dex70, i.e., a 70 kD dextranpolymer substituted with 25 fluorescein hapten groups. The time at whichthe dose of 1 mg of polymer was given was arbitrarily designated day 0on the time scale. The open circle data points are the averages of thedata points shown in FIG. 27 for unsuppressed mice, with standarddeviations and least square fit straight line indicated.

FIG. 30. Cure of anti-fluorescein IgG antibody response by thefluoresceinated dextran polymer FL30-Dex80, i.e., a 80 kD dextranpolymer substituted with 30 fluorescein hapten groups. The time at whichthe dose of 3 mg of polymer was given was arbitrarily designated day 0on the time scale. The open circle data points are the averages of thedata points shown in FIG. 27 for unsuppressed mice, with standarddeviations and least square fit straight line indicated.

FIG. 31. Cure of anti-fluorescein IgG antibody response by thefluoresceinated dextran. ELISA assay results are shown from serum ofmice diluted 100,000 fold. These mice were stimulated at the timesindicated by doses of 10 μg of fluoresceinated ovalbumin absorbed on 1mg of aluminum hydroxide. Cure was by injection of 2 mg of highlyfluoresceinated dextran of average molecular weight approximately 40 kDat the times indicated. Data are from groups of three mice with mean andstandard deviation indicated.

FIG. 32. Similar to FIG. 31, except that mice were stimulated at thetimes indicated by doses of 1 μg of fluoresceinated ovalbumin absorbedon 1 mg of aluminum hydroxide.

FIG. 33. Similar to FIG. 31, except that mice were stimulated at thetimes indicated by doses of 0.1 μg of fluoresceinated ovalbumin absorbedon 1 mg of aluminum hydroxide.

FIG. 34. Reduction by cure treatment of the number of splenocytesproducing anti-FL IgG serum antibodies. The reduction in the populationof antibody-secreting cells is substantial over a wide range ofinitially stimulatory doses of FL-OVA on aluminum hydroxide.

FIG. 35. Percent reduction of anti-fluorescein IgG producing lymphocytesfrom the spleens of mice which had been cured with FL-Dex. The mice hadbeen treated as shown in FIGS. 31-33, with the final cure dose of FL-Dexgiven on day 95 and the spleen cells analyzed on day 125, 30 days later.

FIG. 36. Two separate PCA measurements, using different rats ondifferent days, of the IgE antibody levels of pooled sera from cured anduncured mice, 6 mice per group. Stimulatory doses of 0.1 μg FL-OVA on 1mg aluminum hydroxide were given on days 0, 21 and 71 to all the mice.For the cured mice, cure of the anti-fluorescein IgE serum antibodyresponse was by treatment with 2 mg doses of FL30-Dex80 on days 34 and95.

FIG. 37. The structures of penicillin and the penicilloyl hapten. R is abenzyl group for Penicillin G (benzyl penicillin). In the penicilloylhapten, the internal amide bind of the β-lactam ring is replaced by anamide bind involving a primary amine from the carrier.

FIG. 38. Administration of the suppressive polymer BPO-PA virtuallyabolishes the anti-BPO response (FIG. 38a), while the anti-OA response(FIG. 38b) is unaffected. Not only does the anti-BPO titer remainundetectable for two months, but the mice are tolerized by the BPO-PAand are unresponsive to a “booster” injection given on day 110.

FIG. 39. Supression of ongoing anti-fluorescein IgG response usingvalence restricted scaffolds.

FIG. 40. Serum anti-BSA IgM dose-response for monomeric and polymerizedBSA. CAF1/J mice were injected with 10, 100 or 1000 μg of monomeric (68kD) or carbodiimide cross-linked 70-meric (5000 kD) preparations of BSA.The IgM antibody response was measured by ELISA at day 6 for serumdilutions of 200 fold. Data are the average of 3 mice per point.

FIG. 41. Effect of BSA multiplicity on response. Data similar to thatshown in FIG. 40 for BSA complexes having molecular multiplicity ofapproximately 1, 3, 7, 20 and 70 (BSA monomer has a mass of 68 kD).

FIG. 42. Effect of BSA multiplicity on serum IgG response. Serumanti-BSA IgG antibody levels 14 days after a single injection of BSApolymers of different molecular masses at various doses. Serum wasdiluted 1000 fold, 3 mice averaged per point.

FIG. 43. Serum anti-BSA dose-response for multiple injections ofmonomeric or carbodiimide cross-linked 20-meric BSA. The anti-BSA IgGresponse is shown after three injections given 30 days apart. Serum wasdiluted 4000 fold for ELISA assay.

FIG. 44. Effect of multiple injections on serum anti-BSA IgG response toa BSA “20-mer” given at very low dose. One μg of either monomeric orcarbodiimide cross-linked 20-meric BSA polymer, 1400 kD, was injected ona monthly basis. A total of 5 μg (5 injections) was given. Data areaveraged from 3 mice.

FIG. 45. Serum anti-OVA IgM dose-response 5 days after injection of OVAmonomer or glutaraldehyde cross-linked 150-mer.

FIG. 46. Serum anti-OVA IgG response to multiple injections of monomericor glutaraldehyde cross-linked OVA fractions of different molecularsizes. Serum was diluted 4000 fold for ELISA assay.

FIG. 47. Comparison of serum IgM response generated by monomeric andvarious polymeric sizes of BSA and OVA. Three monthly 1 mq injections ofmonomeric or polymerized BSA or OVA were given in saline. Serum wasdiluted 200 fold.

FIG. 48. Comparison of the anti-fluorescein and anti-BSA serum IgGresponse generated by a fluoresceinated BSA polymer. Samples of a BSApolymer (20-mer) as haptenated with fluorescein isothyocyanate, yieldingpreparations with different amounts of fluorescein per BSA monomer unit.Mice were injected with 4 biweekly doses of 100 μg each in saline, for atotal dose of 400 μg. Serum was assayed for IgG antibodies tofluorescein and to BSA.

FIG. 49. Levels of IgG peptide (GALA)-specific antibodies in serum.

Legend: Mouse #5 ∘—∘; Mouse # ∘———∘; Mouse #7 Δ...Δ; Mouse #8 ▴—.—.—▴;*=buffer injected.

FIG. 50. Prevention of rise in antibody levels by EALA-DEX-84. Cure ∘—∘;control —.; Control 1/19 0.3 ml buffer; Cure EALA DEX-84.

FIG. 51. Specific suppression of 104 response directed towards the 159epitope(s). Bleed after day 77 cure was on day 83; on day 84—bleedd7=d91. Legend: ↑ “Cure” mice were injected with various doses of159₁₀-dex₄₀; control mice were injected with saline. * Control and curedmice were injected intraperitoneally with 10 μg 104-BSA.

FIG. 52. Suppression of anti-histone antibody titers. FIG. 52a isexperimental group, FIG. 52b is control group.

Protocol

Day 1—1, 10, 100 μg I.V. or 100 μg I.P.

Day 3—200 μg I.P.

Day 9—200 μg I.P.

Day 16—200 μg I.P.

Day 23—200 μg I.P.

Legend: mouse # —□— 1; —∘— 2; —□— 3; — — 4; —∘— 5; —□— 6; —▴— 7; —Δ— 8;—□— 9.

FIG. 53. Specificity of suppression of anti-histone responses.

FIG. 53a—Anti-N15-H2B.

FIG. 53b—Anti-ssDNA.

Legend: Experimental=—□—, N=17; Control=—∘—, N=11.

FIG. 54. Activation and inhibition of T-cell interleukin-2 production bysoluble fluorescein polymers. Transfected T-cell line 1B2 was treatedwith phorbol ester, 3 ng per ml, and with various concentrations ofsoluble fluorescein polymers as indicated in the Figure. Afterincubation, supernatant solution was removed and assayed for IL-2 bymeasuring proliferation of an IL-2-dependent cell line, CTLL2.Proliferation of the CTLL2 cells is measured by the incorporation ofradioactive thymidine into cellular DNA. In FIG. 54a, the T-cellresponse to two fluorescein polymers of different molecular weight andvalence were measured at various concentrations. In FIG. 54b, forvarious indicated concentrations of the stimulatory polymer, theinhibitory polymer was added at four concentrations: none (opensquares); 0.48 μg/ml (closed triangles); 4.8 μ/ml (X symbols); and 15μg/ml (closed squares).

FIG. 55. Activation and inhibition of intracellular calcium flux inT-cells by soluble fluorescein polymers. Transfected T-cells were loadedwith the calcium sensitive fluorescent dye, Indo-1 AM (Molecular Probes,Eugene, Oreg.). Fluorescence emission at two wavelengths, 405 and 480nm, was determined upon excitation at 355 nm for individual cells, usinga Coulter MDADS flow cytometer. In the Figure, each dot represents thecalcium concentration in a single cell at some instant in time, withtime shown in units of 16 seconds on the abscissa. The transfected cellswere analyzed for 20 seconds and then various fluorescein polymers wereadded in the complete absence of phorbol ester or accessory cells.Substantial intracellular calcium concentration rises in at least 10% ofthe cells were seen when the cells were treated with the stimulatorypolymer. FL50-Fic150, at concentrations of 38 μg/ml (a) and 3.8 μg/ml(b), but less calcium flux at 380 μg/ml (c). However, the inhibitorypolymer, FL11-Fic46, did not induce any substantial calcium flux at anymeasured dose, but caused substantial inhibitory effect (d and e).Stimulatory polymer, FL50-Fic150, 38 μg/ml (d) and 3.8 μg/ml (e), wasadded after a short incubation of the cells with inhibitory polymer. Inboth cases the calcium flux induced by the stimulatory polymer is almosteliminate.

DESCRIPTION OF THE INVENTION

The method of the invention comprises administering to a subjectsuffering from an undesired immune response an effective amount of anon-immunogenic material which carries a number of antigenic domains(i.e., “epitopes” or “haptens”) which correspond to the antigen, e.g.the allergen or self-antigen which causes the allergy or autoimmunedisease responsible for the undesired response. The haptens or epitopesbind to cell antigen receptors specific for the indicated haptens orepitopes and, provided the hapten or epitope number is sufficient andthe carrier size is below an ascertainable threshold limit so as toavoid the formation of a stimulatory cluster of antigen receptors, theadministered material serves to suppress or abolish the specific immuneresponse. The administered material specifically suppresses the immuneresponse to the allergen or self-antigen, without compromising ordamaging the general immune competence of the body.

The disclosure of application Ser. No. 07/248,293 includes a descriptionof constructs comprising size fractionated linear polyacrylamidechemically modified to accept DNP groups as epitopes. These conjugatescan be organized into groups based on the size (molecular weight) of thebackbone polymer and hapten number (number of DNP groups per averagemolecular weight polymer for a given group). The combination of thesetwo scaler quantities makes it possible to determine the role of haptendensity as a separate variable. Based on the data obtained using theseconstructs in both in vitro and in vivo models of immune function,certain “rules” governing B-cell activation by antigen have beenelucidated and used to control the T-cell independent immune response onan antigen specific basis. These rules and their use in effecting anantigen specific alteration in immune function are included inapplication Ser. No. 07/248,293.

Application Ser. No. 07/354,710 included further exemplification insupport of the application of these rules to include a variety ofbackbones or scaffolds and haptens, thus further documenting the“universality” of the rules elucidated in the original filing as theyapply, particularly, to T-cell independent immune system activities(operationally defined as IgM production). The present disclosureincludes specific exemplification which makes clear the applicability ofthese selfsame rules to a spectrum of immune function, including T-celldependent antibody production (operationally defined as the productionof IgG and IgE) and T-cell activity as well.

The Examples that follow include the exemplification from the parentcases and further exemplification of complex constructs involvingantigens of greater diversity than simple small molecular weight haptenssuch as DNP and fluorescein. The biophysical and biochemicalconsiderations that need to be taken into account when designing theseconstructs are set forth below. These include the chemistry of synthesisof the constructs and preferred methods of characterizing the finalproducts so as to optimize fidelity to and compliance with the primaryprinciples governing valence and size that constitute the operationalunderpinnings of the invention as disclosed in this series ofapplications.

For a construct (conjugate) to be non-stimulatory, and hence“suppressive” or tolerogenic in nature, it must meet one or both of thefollowing criteria:

i) The “valence” of the conjugate (operationally defined as the numberof “discrete antigenically recognizable moieties” per finalmacromolecular construct) must be less than the Immunon model thresholdnumber (generally, less than 20). As noted above, these moieties can besimple haptens or more complex peptides or proteins. It will beappreciated that each of these moieties may have multiple “antigenicfacades”, but for any given B-cell, capable of recognizing the moiety,it will have one and only one discrete binding region recognized by oneimmunoglobulin receptor of that particular B-cell even though otherB-cells may recognize other regions of the moiety in question. Specialcases such as peptides or proteins containing multiple identical peptidesequences (such as some of the sequences found in certain malariaproteins or in proteins such as hemoglobin which has repeated subunits)or carbohydrates with regularly repeating series of sugar residues (suchas in bacterial polysaccharides) are considered as containing multiple“discrete antigenically recognized moieties” for purposes of definingvalence; and/or

ii) The size of the final construct must be smaller than the minimumsize necessary for spanning the cluster of receptors defining the“Immunon”. It will be appreciated that the effective size will be afunction of a number of independent parameters including: geometry ofthe backbone or scaffold (linear, branched, globular, radial, etc.), thephysical nature of the backbone (flexible, rigid, “articulated”, etc.),the hydrophilicity or hydrophobicity of the backbone, the electrostaticnature of the conjugate (the sum of charges on both the backbone and thearrayed moiety described above), and the size, geometry and physicalmake-up of the moiety itself.

It will be appreciated that the optimum number and spacing for aparticular hapten or epitope as well as the carrier size can bedetermined without undue experimentation by simple tests on experimentalanimals such as mice, rats, rabbits or guinea pigs, using the selectedscaffold material and antigenically recognizable moiety (see theabove-referenced 1976 paper).

I. Preparation of Immunosuppressive Constructs

Constructs suitable for use in the present invention can be producedusing known means. Preferably, the production method used is one whichminimizes the possibility of polymerization as well as cross-linkingbetween the individual molecules. In addition, the production method is,preferably, chosen such that only one potential reactive site perarrayed moiety is available so that the orientation of the moiety to thebackbone can be controlled. Resulting construct preparations are,advantageously, characterized prior to use to ensure that they aresubstantially free from high molecular weight, potentially stimulatorymolecules. The use of valence restricted scaffolds of defined chemistryis preferred in order to optimize reproducibility of the resultingconstruct.

Construct Design and Analysis

A. General Chemistry Considerations

The fundamental concept underlying the technology upon which Applicants'invention is based is that the immune system interacts with its externalmilieu by the recognition of antigenic arrays of epitopes or haptens.From the biophysical or biochemical perspective, epitopes or haptens areno different from any other receptor ligand, and the solubleimmunoglobulin molecules and their membrane bound relatives (such as theT-cell receptor, the B-cell receptor, etc.) are no different than anyother protein receptor molecule in other biological systems. Thedifference lies not with the individual receptor-ligand interaction butwith the mechanism of “information transfer” that occurs after theligand is bound by the receptor. While the individual membrane boundreceptors in the immune system can and will bind monovalent ligands, thefunctional interaction for this type of ligand-receptor interaction isthe time stable clustering of receptors into discrete units termed“immunons”. This phenomenon differs qualitatively from receptor-ligandinteractions in other biological systems wherein each individual bindingevent has functional importance. (For example: when a singleneurotransmitter molecule binds to its receptor on the post-synapticmembrane, a change in membrane potential can be measured.) While immunonformation depends on the binding of individual ligands (epitopes orhaptens) with individual receptors, the immunon itself is dependent onthe biophysical characteristics of the entire array and not thesummation of individual binding events.

A corollary to this discussion of receptor-ligand interaction in theimmune system as compared to receptor-ligand interactions in otherbiological systems is the concept of subthreshold and superthresholdarrays being immunologic “antagonists” and “agonists”, respectively. Fora classical pharmacologic antagonist to have acceptable potency it mustbind to the receptor molecule with approximately the same degree ofaffinity as an agonist but in a “nonproductive” manner. That is, it mustbind but not activate the secondary events caused by agonist binding.Since the functional event in the immune system is immunon formation andnot individual receptor-ligand interactions, the corollary to an“antagonist ligand” is the “antagonist array” that can aggregatereceptors in nonproductive clusters thereby preventing the formation ofan immunon by an “agonist array”. Using this concept of productive andnonproductive receptor clustering, immunologic agonists can be viewed as“superthreshold arrays” that can bind with a number of receptors thatmeets or exceeds the minimum necessary for immunon formation andimmunologic antagonists can be viewed as “subthreshold arrays” thatcannot induce immunon formation but can still occupy multiple receptorssimultaneously with approximately the same degree of aggregate avidityas the superthreshold (agonist) array.

Finally, it should be apparent that if the immunon concept (mechanism)is operationally enabling, the specific chemistry of the array isunimportant as long as the biophysical rules of receptor clustering aremet and the ligands being arrayed can be recognized by the intendedpopulations of receptors. For example, the targeted cell populationsshould not be sensitive to the exact nature of the scaffold used as longas the array is capable of interacting with the requisite number ofreceptors. The desired outcome can be achieved with a myriad differentconstructs as long as the principles of valence and/or size aremaintained with fidelity. As a result, it is just as important tocontrol the chemistry of the scaffold or backbone upon which theantigenic array is based as it is to identify and synthesize theappropriate ligand. One skilled in the art will appreciate that it isalso important to confirm the integrity and composition of the finalconstruct used before it is introduced into a biological system.

For example, if an immunon consists of eight receptors brought into acluster, subthreshold clusters can be achieved by presenting the immunesystem with rigorously defined “valence restricted” antagonist arrayswherein the number of ligands is restricted to an integral number lessthan eight. In a naive animal, such arrays will be non-stimulatory andwill prevent an antibody response to the epitope in question fromdeveloping. In an animal in which an immune response directed againstthe same antigen is already established, these constructs will act ascompetitive inhibitors to that response, i.e. they will be suppressivein nature.

On the other hand, an immunon must have a finite minimum size which isdetermined by the maximum packing density that the requisite number ofantigen receptors can achieve on the surface of the lymphocyte. Arraysthat cannot cover this minimum area (size restricted antagonist arrays)can be expected to be both non-stimulatory and suppressive no matter howmany binding sites they may have. For small ligands such as DNP,fluorescein, or small peptides, the major determinant for array size isthe scaffold, hence the size limit (preferably, less than approximately150,000 daltons). But, for more complex ligands such as largerpolypeptides, nucleic acids, or even proteins, the “ligands” themselvesmay be the controlling element with regard to the size of the finalconstruct. In this case, even valence restricted constructs may exceedthe nominal “size” criteria established for smaller epitopes. In thiscase, valence considerations will predominate over size considerationswith respect to how the immune system will respond. An immunon cannot beexpected to form if the array being introduced into the system has asubthreshold number of receptor binding sites no matter how big thearray is in absolute terms.

In either case (size restricted or valence restricted antagonistarrays), many different chemistries can achieve the same outcome.Addressed below are three technical points important to the presentinvention. The first point is the preparation of the backbone scaffoldso that the ligands can be covalently attached thereto in a controlledand selective manner. The second point is the preparation of the ligandsthemselves so that they can be attached to the scaffolds in a specificorientation. The third point is the formation and characterization ofthe final conjugate prior to its introduction into a biological system.

B. Scaffolds

Many different biologically acceptable carriers or scaffolds can be usedto effect the desired outcome with respect to the formation of agonistor antagonist arrays. Some are capable of being used without selectivechemical modification. The only significant restriction placed on thesescaffolds is that they be freely soluble in physiologically acceptableaqueous buffers when the final constructs are prepared. The scaffold canbe used to provide the necessary solubility for the final product eventhough the ligand being conjugated to the backbone is relativelyinsoluble. Alternatively, if the ligand in question has undesirablecharge characteristics (e.g., cationicity) the scaffold can be used tocounterbalance these characteristics so that the final product fallswithin preferred tolerances.

For general conjugation reactions, introduction of, for example, primaryamines onto the scaffold provides a functional group capable ofaccepting multiple chemical modifications or manipulations that can beachieved using mild conditions in aqueous solutions. One skilled in theart will appreciate, however, that alternative chemistries can also beemployed for these types of reactions. Set forth below is arepresentative sampling of the chemistries that can be employed toprovide the necessary scaffolds for conjugation. Table 1 summarizes thedifferent biophysical characteristics of certain scaffolds (see alsoExample 1) and underscores the generality of the concept.

TABLE 1 Molecular Characteristics of Polymer Molecules Carrier PolymerCarrier Composition Dextran Bacterial Predominantly polysaccharidelinear, of glucose somewhat subunits branched homopolymer PolyacrylamideSynthetic Linear polyethylene homopolymer, polymer uncharged FicollPolysaccharide Three- synthesized dimensional & from sucrose highlycrosslinked Carboxymethyl- Carboxy- Linear, cellulose methylatednegatively plant charged polyglucose homopolymer Polyvinyl SyntheticLinear alcohol polyethylene uncharged polymer homopolymer Poly (D-GLU/D-Synthetic Linear LYS) polymer of variably “unnatural” D- charged randomamino acids polymer of D- glumatic acid and D-lysine Protein NaturallySize Oligomer occurring fractionated Serum soluble oligomer of Albuminprotein crosslinked Chicken globular Ovalbumin proteins Serum Immuno-globulin

Preferably, the materials used are subjected to analytical and, ifnecessary, preparative sizing techniques (for example, size exclusiongel chromatography or ultrafiltration) to ensure homogeneity andrelatively narrow mass distributions both before and after modification.In addition, independent verification of mass (for example, by laserlight scattering and/or equilibrium ultracentrifugation) can also beperformed on these materials both before and after modification so as toassure that the scaffolds used are within the size tolerances needed foreither agonist or antagonist array formation.

C. Epitopes

The chemistry necessary for modifying the desired ligand for attachmentto the chosen scaffold is described generally below. Again, the specificchemistry employed can be modified or changed in a variety of ways. Oneskilled in the art will recognize that the details set forth herein arebut examples of the types of chemistry available for producing theconstructs to which the present invention relates.

1. Low molecular weight haptens

The low molecular weight haptens specifically described herein werepresent in a form that could react directly with the available freeamines on the scaffolds utilized without modification. For example, thefluorescein derivatives were formed using fluorescein isothiocyanatewhich rapidly reacts with available amines forming a stable thiourealinkage. Those skilled in the art will recognize that other smallmolecular weight haptens can also be employed using known chemicalprotocols.

2. Peptides

Peptides identified for use as a ligand can be modified so that they canbe successfully arrayed and yet still be recognized by the immune systemin the desired fashion. Naturally occurring peptides or proteins havethree types of amino acid side chain moieties that can be readily usedas functional groups with which to tether the peptide to the desiredscaffold. These groups are: amines, as represented by the epsilon aminogroup of lysine and the N-terminal alpha amino group; carboxyls, asrepresented by the side chain carboxyl groups of aspartic or glutamicacid and the C-terminal alpha carboxyl; and the sulfhydryl group ofcysteine. As one skilled in the art will appreciate, the side chains ofglutamic acid, aspartic acid and lysine are frequently found on theexternal surface of proteins and, as a result are commonly involved inantigen-immune system interactions. Peptides containing a combination ofmore than one amino and/or carboxyl group require particular attentionin terms of orientation specific and controlled chemistry. In addition,the potential for conjugate polymerization that exists when theseresidues are used for array formation must be addressed.

Use of the free sulfhydryl group of cysteine has a number of significantadvantages. First, in most cases in biology, cysteine does not exist asa free sulfhydryl. As a result, it is rarely involved in antigen-immunesystem interactions. Second, there is a wealth of chemistry that takesadvantage of sulfhydryls to the exclusion of any other reactive groupcommonly found in biology. Further, cysteine, as a naturally occurringamino acid, can be incorporated into recombinantly synthesized proteins.For these and other reasons discussed in more detail below, sulfhydrylchemistry is the preferred system for conjugating peptides to variousscaffolds.

For all of the peptides specifically described herein, standard solidphase peptide synthesis techniques have been employed, the specifics ofwhich are described in Example 9. When the peptide in question isconjugated to a protein, the issue of the ability to unambiguouslyquantitate the amount of peptide linked to the carrier must beaddressed. In order to prevent significant steric factors frominterfering with ligand binding, spacers of various kinds can beincorporated into the ligand in question.

Various “unnatural” omega amino carboxylic acids, such as epsilon aminocaproic acid or delta amino valeric acid, can be used as spacers betweenthe ligand in question and the cysteine (or cysteamine—see below) usedto link the peptide to the scaffold. These amino acids have uniqueanalytical characteristics when subjected to standard amino acidanalyses and can be used to quantitate peptide “valence” as well asallow for a flexible linker between the peptide in question and thescaffold.

3. Proteins

Proteins provide for significantly different considerations with respectto the immune response generated to these types of antigens. Theseinclude multiple different antigenic epitopes per protein monomer aswell as different types of epitopes (sequential, linear conformational,and discontinuous conformational epitopes). In order to deal with theseissues, three categorically different approaches to protein antigens,their epitopic representation, their synthesis and their deployment inagonist or antagonist arrays, need to be taken into consideration. Thefirst of these issues is the “mapping” of a protein's antigenic facadewith smaller peptide or modified peptide based ligands. The second isthe use of oligomeric constructs made up of the whole proteins ordomains of larger proteins either crosslinked to themselves or to ascaffold. And, the third is the generation of “mimotopes” which canmimic the antigenic structure of protein epitopes but which bear littleor no compositional similarity to the naturally occurring antigen. Theseapproaches are described below in Example 10.

4. Carbohydrates

Set forth below are specific methods of covalently assembling molecules,that is, of coupling polysaccharides, oligosaccharides, sugars,glycoproteins or other materials through their reducing end groups toform larger molecular arrays. Alternatively, the chemistry describedbelow can be used to attach polysaccharides, oligosaccharides,glycolipids or glycoproteins as haptens to a different molecularscaffold.

Reactive end groups, amino or sulfhydryl, suitable for coupling to othermolecules can be produced in high yield by the following procedureswhich make use of the formation and selective reduction of intermediateSchiff bases:

Primary aliphatic amino groups—The saccharide material is reacted (forexample, for 18 hours, at pH near 5) with ethylenediaminedihydrochloride (concentration, for example, 0.1-1.0 M) (or other smalldiamine, NH2-(CH2)n-NH2, where n is a small number 2 or greater), in thepresence of 0.01 M sodium cyanoborohydride (concentration, for example0.01 M). Upon removal of reagents by dialysis or a desalting column,substantially complete reaction of reducing end groups is obtained, withformation of terminal primary amino groups suitable for subsequentcoupling reactions.

Sulfhydryl groups—The saccharide material is reacted, as above,cysteamine dihydrochloride as the diamine (concentration, for example,with 0.1-1.0 M). Upon completion of reaction, the resulting derivativedisulfide can be readily reduced with standard disulfide reducingagents, such as Cleland's reagent, to yield free terminal sulfhydrylgroups suitable for subsequent coupling reactions.

5. Nucleic Acids

In at least one application (the treatment of systemic lupuserythematosus), the antigen is known to be a nucleic acid—doublestranded DNA. In a series of experiments designed to assess the minimumsize of unmodified double stranded DNA needed for successful receptorbinding it was found that approximately 40 base pairs were needed for100% receptor binding. This requirement may be different if the doublehelix is covalently crosslinked instead of relying solely on thehydrogen bonding of the base pairs for stabilization.

Naturally occurring DNA, synthetic DNA or modified DNA containingphosphorothioates as opposed to naturally occurring phosphate linkagescan be used to produce a successful ligand. Example 11 includes adescription of the types of chemistries that can be employed to producethe desired epitopes possessing the necessary functional groups.forcovalent attachment to the appropriate scaffold.

D. Conjugates

The final steps in the preparation of a conjugate suitable for use inthe method to which the invention relates is the assembly of the desiredarray from the appropriate scaffolds and ligands and the confirmationthat the final material is, in fact, what it is intended to be.Characterization of the final constructs is an important part of thepreparation and use of these materials (see Example 12).

II. Utilization of Constructs to Suppress T-Cell Dependent ImmuneResponses

As indicated above, the earlier filed applications of the present seriesrelate, in large part, to the suppression of T-cell independentresponses by constructs comprising size restricted backbones and smallmolecular weight haptens (such as DNP and fluorescein). Data presentedin the Examples that follow demonstrate that the same type ofsuppression can be obtained with more complex responses involving T-celldependent antibody production, represented by IgG and IgE.

As will be evident from the Examples that follow, rapid and completeobliteration of a hapten-specific antibody response (represented by IgGproduction) can be effected by administration to animals of asuppressive construct specific for the hapten, prepared as describedherein. The data indicate that suppression occurs at the cellular level.Clinically important antibody responses to extrinsic allergens (bothsmall chemical entities and complex epitopes) represented by IgEproduction can be completely suppressed by constructs meeting thevalence and size criteria set forth above. In addition, constructs ofthe present invention can be used to suppress autoimmune responses.

It will be appreciated from the results set forth in the Examples thatfollow that the methodology presented here, as well as in the earlierfiled applications, is broadly applicable to T-dependent antibodyresponses to small molecular weight haptens as well as to complexantigens (peptides, proteins lipoproteins, glycoproteins, nucleic acids,carbohydrates, lipids and glycolipids). The principles involved are notdependent on the use of specific backbones, or scaffolds. Polymershaving vastly different chemistry (dextran, polyacrylamide, Ficoll,etc.) can be used. Further, and as indicated above, the technology isapplicable to T-dependent antibody responses of all classes to any andall extrinsic (allergic) or intrinsic (autoimmune) antigens that complywith the rules of the Immunon model of antigen recognition.

The following non-limiting Examples describe certain aspects of theinvention in greater detail.

EXAMPLES Example 1

Scaffold Synthesis

A. Dextran

Dextran can be considered a “prototypical” scaffold for a number ofreasons: 1) it is freely soluble in aqueous buffers, 2) it can bereadily modified using “off the shelf” chemistry, 3) it has been used inhumans in gram quantities as a plasma expander with no significanttoxicities, 4) it is available in roughly size-fractionated bulkquantities at low cost and 5) there are no known mammalian dextranases.The latter point is particularly important since one of the primaryconsiderations of this technology is that the arrays be metabolicallystable so that the desired outcome can be effected in an experimentalanimal or human.

Representative examples using different chemistries are presented here.One skilled in the art will appreciate that chemistries used to modifydextran can also be extrapolated to other systems.

a. Basic “dexamine” and GMB-dexamine

The chemistry used to activate dextran such that covalent attachment ofa peptide can occur is shown in FIG. 1. Dextran of various molecularweights (1) was first carboxymethylated with chloroacetic acid(ClCH₂CO₂H) at glucosyl 2′-, 3′- or 4′-hydroxyl positions to yield thecorresponding carboxymethyl-dextran (2). Conversion into dexamine (3)was then accomplished by reaction (of carboxymethyl-dextran) withethylene diamine (NH₂CH₂CH₂NH₂) in a water soluble carbodiimide(EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride)-mediated coupling reaction. The presence of free aminogroups in dexamine (3) allows measurement of the “amine substitutiondensity” using standard quantitative analytical chemistry techniques.Expressed as the number of μmoles of amine present per milligram ofdexamine (3), the value for amine density represents the theoreticalmaximum substitution density for any particular peptide. Dexamine (3)used in the Examples described herein was found routinely to containapproximately one primary amino group (—NH₂) for every five glucosylresidues (or 1000 g/mole) present in the dexamine (3) sample.

Derivatization of the dexamine (3) amino groups with theheterobifunctional acylating agent: gamma-maleimido n-butyric acidN-hydroxysuccinimide ester (GMBS) then gave the activated or“conjugatable” form of dextran: gamma-maleimido n-butyryl dexamine(GMB-dexamine, 4). It is the maleimide functional group withinGMB-dexamine (4) that reacts with (i.e. conjugates with) the sulfhydrylgroup of a cysteine-containing peptide to generate a peptide-dextranconjugate.

The dextran (1) used in the preparation of the peptide-dextranconjugates was a size-fractionated, average molecular weight polymer.Chloroacetic acid and ethylene diamine were purchased from AldrichChemical Co. 1-Ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride(EDC) and gamma-maleimido n-butyric acid N-hydroxysuccinimide ester(GMBS) were purchased from Sigma Chemical Co. Trinitrobenzensulfonicacid (TNBS) was obtained from Pierce Chemical Co. Phosphate-bufferedsaline (PBS; 150 mM NaCl, 10 mM phosphate pH 7.3-7.4), used in thepreparation of GMB-dexamine (4), was prepared fresh (for each day's setof reactions) from an autoclaved 10×PBS stock solution and autoclavedwater with subsequent filtering through a 0.2 μm filter. (Preparation ofPBS in this manner is important to the production of conjugates that aredevoid of undesirable contaminants). Dialysis tubing (6,000-8,000 mwco)was obtained from Spectrum Medical Industries, Inc.

Purification of dextran (1) and molecular weight measurement of dextran(1) or dexamine (3) samples were carried out by size exclusionchromatography followed by equilibrium ultracentrifugation, and/or laserlight scattering analysis.

Carboxymethyl-dextran (2) was produced from dextran as follows: Sodiumhydroxide (675 mmole, 135 mL of 5 M NaOH) was added to 0.3 L of waterand the resulting solution chilled in an ice-water bath (0° C.) withstirring. Chloroacetic acid (64.4 g, 685 mmole) was then added andstirring continued at 0° C. until complete dissolution occurred. Theresulting solution was allowed to warm to room temperature and the pHwas adjusted to ca. 7 by the addition of either NaOH or chloroaceticacid. After being diluted to 0.5 L total volume, 185 mL of thechloroacetate solution was added to the dextran sample (0.143 mmole) andthe carboxymethylation reaction initiated by the addition of 50 mL of 10M NaOH (500 mmole). The reaction mixture was diluted to 250 mL totalvolume and carboxymethylation allowed to proceed for 20 hours at 37° C.The reaction was then terminated by adjusting the solution pH to ca. 7with 6 M HCl. After being allowed to cool to room temperature, thereaction mixture was dialyzed for 5 days against water (two waterchanges per day) and the resulting carboxymethyl-dextran (2) isolated bylyophilization.

Dexamine (3) was produced from carboxymethyl-dextran (2) as follows:(0.143 mmole carboxymethyl-dextran (2) reaction scale):Carboxymethyl-dextran (2) was dissolved in 300 mL of water and ethylenediamine was added (45 g, 750 mmole). The resulting solution was stirredat room temperature and the pH adjusted to ca. 5 with 1 M HCl.1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 4 g,20.86 mmole) was then added portionwise over a 10 to 20 minute periodwith continuous stirring. The pH of the reaction mixture was checkedevery 15 minutes and maintained near pH 5 (via the addition of 1 M HCl)for 4 hours. Extensive dialysis was then carried out as follows:overnight against 30% AcOH, 24 hours against water (2 water changes),overnight against 30% AcOH, 24 hours against water (2 water changes),overnight against 1 M NaCl and then 48 hours against water (2 waterchanges per day). The resulting dexamine (3) was isolated bylyophilization.

Measurement of (dex)amine content (i.e. amine substitution density) wascarried out as follows: Dexamine (3) was dissolved in 1 mL of 0.1 Msodium tetraborate buffer (pH 9.3) to give a solution concentration of1-2 mg/mL. Freshly-prepared trinitrobenzenesulfonic acid (TNBS, 25 μL ofa 30 mM solution in sodium tetraborate buffer) was added and theresulting (vortexed) reaction mixture stored in the dark for 2 hours atroom temperature. The yellow-colored solution was then read against areagent blank at 366 nm. Standards of 1 mM lysine, 2 mM glycine or 2 mMaminobutyric acid were prepared and a standard curve generated fromvarious aliquots diluted to 1 mL total volume with buffer.

GMB (gamma-maleimido n-butyryl)-dexamine (4) was produced from dexamine(3) as follows: Dexamine (produced as described above) was dissolved inphosphate-buffered saline (PBS, pH 7.5) and stirred at room temperatureto give a solution concentration of 5-10 mg/mL. A five-fold molar excessof GMBS (gamma-maleimido n-butyric acid N-hydroxysuccinimide ester)relative to (dex)amine content was then dissolved in dry tetrahydrofuran(THF, stored over 4 Angstrom molecular sieves) such that a GMBSconcentration of ca. 50 mg/mL was achieved. The GMBS/THF solution wasadded dropwise to the stirring dexamine (3) solution and the acylationreaction allowed to proceed for 30 minutes at room temperature with thesolution pH being maintained at 7.1-7.5 by the dropwise addition of 1 mMNaOH. GMB-dexamine (4) was then separated from excess GMBS by gelfiltration of the reaction mixture on a 15 mm×30 cm Sephadex G-25 columnequilibrated in PBS (pH 7.5). The column effluent was monitored at 280nm with a Pharmacia Dual Path Monitor UV-2. Column fractions containingGMB-dexamine (4) were combined and set aside pending the availability ofa reduced cysteine (Cys)-containing peptide.

b. Alternative Neutral, Anionic and Cationic “Dexamines”

As mentioned above, under certain circumstances the scaffold will needto compensate for an undesirable charge profile of the ligand inquestion. Described below are means by which dextran-based scaffolds canbe made to have neutral, anionic or cationic characteristics using amodification of the fundamental dextran chemistry described above.

For the preparation of neutral scaffolds, the first modification ofdextran (1) to carboxymethyldextran (2) is identical to that describedabove. At this point, however, two different amino amides (β-Ala-CONH₂or L-Gln-CONHCH₃) can be condensed with carboxymethyl dextran to formtwo different types of “dexamides” which will ultimately be convertedinto charge neutral dexamines described in FIG. 2.

For the preparation of anionic scaffolds, an alternative precursordexamide is needed. These can be formed by the condensation of eitherL-Asn or L-Gln with dextran according to the following protocol:

4-Nitrophenyl chloroformate (685 mg, 3.4 mmole) was added to a solutionof 1 g dextran (1) (13.87 μmole) in 60 mL of a dry DMSO-pyridine mixture(1:1, v/v) at 0° C. in an ice-water bath. To this solution was added 76mg of 4-(N,N-dimethylamino)pyridine (6.22 mmole). The reaction mixturewas stirred at 0° C. for 4 hours and then a fifty-fold molar excess ofeither L-Asn or L-Gln was added. The reaction mixture was allowed towarm slowly to room temperature and then stirring was continued for anadditional 48 hours. The dexamide was precipitated with an excess of dryethanol/ethyl ether (8:2, v/v) and then dried in vacuo. The driedmaterial was dissolved in water, dialyzed for 3 days against water andthen lyophilized (1×).

Once the desired dexamide is prepared it can be converted into itscorresponding neutral or anionic dexamine by first dissolving thedexamide in 50% aqueous acetonitrile and stirring gently at roomtemperature. To this solution, a several fold molar excess (relative tothe calculated amide content) of iodobenzene diacetate is added and thereaction mixture is stirred overnight. (Iodobenzene diacetate willstoichiometrically convert one equivalent of a primary amide into thecorresponding primary amine.) The resulting dexamine can be purifiedfrom the other reaction products by size exclusion chromatography,vacuum concentration and then lyophilization from water. FIG. 3illustrates the complete conversion of dextran to anionically chargeddexamine.

While not as useful as dexamine possessing an anionic character,dexamine with a net positive charge (following conjugation) might befound useful in certain limited cases. Preparation of this material canbe carried out with ethylene diamine-containing dexamine as follows:Acylation of the (dex)amine (4) groups with N ^(α)-Npys-L-Arg-OSuresults in the incorporation of one equivalent of positive charge forevery equivalent of acylated (dex)amine (4). Subsequent removal of the N^(α)-Npys group (with (n-Bu)₃P) liberates a free α-amino group which canbe acylated further with GMBS or converted directly into the maleimidefunctional group via N-methoxycarbonyl maleimide. Longer and shorterhomologs of Arg can be used in an analogous fashion (e.g. Homoarg). (SeeFIG. 4)

B. Polyacrylamide and Poly(acrylamine-acrylic acid)

Linear polyacrylamide was synthesized from the monomer is aqueoussolution, giving polymer preparations with average molecular massesvarying from 20,000 to 500,000 kDa, as determined by the methods ofequilibrium ultracentrifugation, high pressure liquid chromatography(HPLC), size-exclusion gel chromatography (SEC) and laser lightscattering. Preparations were size fractionated on appropriate gelfiltration chromatography columns, Sepharose CL-2B, CL-4B or CL-6B,Pharmacia, to yield center cuts with relatively narrow molecular massdistributions, as measured by HPLC.

Usually, such polyacrylamide preparations were chemically substitutedwith amino groups to prepare them for later coupling with haptenreagents. This was accomplished by first hydrolyzing carboxamide groupsfor varying times, in a carbonate-bicarbonate buffer, to produce aseries of preparations with differing carboxyl group content. Suchcarboxylated polymers were then coupled at their carboxyl groups toethylenediamine by the action of water-soluble carbodiimide, generatingan amide bond and a free, amino group in place of each carboxyl groupundergoing reaction. Hapten was subsequently coupled to the resultingamino-substituted polymer by chemical substitution at the amino groups.

For a detailed description of the methods used see: Dintzis, et al(Proc. Nat'l. Acad. Sci., U.S.A., 73:3671-3675 (1976) and Inman, et al(Biochemistry 8, 4074-4082 (1989)).

In order to provide an alternative to neutral linear polyacrylamide andto provide another carrier with anionic characteristics,poly(acrylamine-acrylic acid) was synthesized using a modification ofthe iodobenzene diacetate reaction described above with commerciallyavailable size-fractionated random copolymers of acrylamide acrylicacid. Specifically, I,I-bis(trifluoroacetoxy)iodobenzene is dissolved inDMF to which an equal volume of water is slowly added with continuousstirring. The size fractionated polymer is then added to the reactionmixture and stirred overnight at room temperature. It is thentransferred to a separatory funnel, washed with water equivalent tothree times the volume of the reaction mixture, and extracted withdiethyl ether (4 extractions with 4×volume). The final aqueous layer isthen vacuum concentrated and the residue redissolved in water anddialyzed against water for several days. The dialyzed product is thenfiltered and lyophilized to yield a fluffy, white solid.

Because one equivalent of I,I-bis(trifluoroacetoxy)iodobenzenestoichiometrically converts one equivalent of a primary amide into thecorresponding primary amine, it is possible to vary the aminesubstitution density of the final polymer product. Furthermore, oneskilled in the art will appreciate that the same procedure can beemployed for any acrylamide containing polymer regardless of the acrylicacid content. The conversion of poly(acrylamide-acrylic acid) into thecorresponding amine-containing polymer is shown in FIG. 5.

C. Ficoll

Ficolls, like dextrans, are polysaccharide polymers that have freehydroxyls that can be modified using the same chemistry as described forthe conversion of dextran to the various dexamines (neutral, anionic,and cationic). In addition, bulk quantities of these materials areavailable from commercial sources in various molecular weight ranges.The difference between dextran based scaffolds and Ficoll basedscaffolds is the fact that Ficolls are more globular or “threedimensional” in nature while dextrans are more linear and branched. Ascan be seen from the data presented in the Examples that follow, thisdifference does not seem to carry with it significant functionalconsequences with respect to the production or use of agonist orantagonist arrays.

D. Carboxymethyl cellulose

Again, carboxymethyl cellulose is very similar to both Ficoll anddextran from the biochemical perspective. This polymer, however, has anet anionic character from the beginning and, as a result, can be usedto produce anionic scaffolds. As will be appreciated, the chemistryneeded to modify this polymer is essentially the same as that fordextran once it has been carboxymethylated.

E. Polyvinyl alcohol

While polyvinyl alcohol is not carbohydrate based, it does possess freehydroxyl moieties that can be modified by the same chemistry asdescribed above for dextran.

F. Poly(D-Glu/D-Lys)

Poly(D-Glu/D-Lys) is a random, linear co-polymer of D-glutamic acid andD-lysine that can be purchases from commercial sources in molecularweight ranges below 100,000 daltons and with an approximate compositionof 40% D=lysine and 60% D-glutamic Acid. Higher molecular weightscaffolds can be produced by the introduction of various water solublecrosslinking agents such as water soluble carbodiimides at variousconcentrations. The resulting crosslinked material can then be subjectedto the same type of size exclusion chromatography and molecular massanalysis as that for the other polymers described above. As the polymeralready possesses free primary amines derived from the epsilon aminogroups of the D-lysine residues, no further modification is necessaryfor these constructs to accept linking groups such as GMBS. It will beappreciated, however, that the ability to control overall charge islimited with this type of preparation.

G. Proteins

Proteins or other polypeptides behave in many respects like thepoly(D-Glu/D-Lys) copolymer with respect to the availability of bothcarboxyl and amino groups for chemical modification. Importantadvantages that proteins offer are set forth below.

First, proteins can be crosslinked and fractionated with respect to sizein a manner similar to the crosslinking and separation of the poly(D-Glu/D-Lys) described above. The fractions can be effectivelysegregated into what would be the equivalent to valence restrictedoligomers (dimers, trimers, etc.). As a result, these constructs can beused as agonist and/or antagonist arrays without further modification.

Second, recombinant DNA/protein engineering technologies have evolved tothe point that fusion proteins made up of a core “scaffold” withrecombinantly produced oligomeric representations of other proteins orprotein domains can be constructed. Again, the final product can beformulated to represent valence restricted arrays of the desired“epitope or ligand” just as if they had been chemically crosslinked orconjugated to a valence restricted carrier.

Finally, streptavidin has a relatively unique structure that can be usedto form tetrameric arrays by the introduction of a biotin moiety ontothe desired ligand. Streptavidin has four binding sites for biotin thathave such high affinity for this moiety that once bound are essentiallythe same as a covalent linkage. In addition, streptavidin is freelysoluble and has an isoelectric point near neutrality so that undesirablecharge characteristics can be avoided.

H. “Point Source” valence restricted scaffolds

The majority of the data disclosed herein has been generated usingsize-restricted scaffolds. As mentioned above, an alternative approachis to use valence-restricted scaffolds for producing agonist orantagonist arrays. Illustrated below are a number of valence-restrictedor “point source” scaffolds that can be utilized for these purposes. Oneskilled in the art will realize that these scaffolds are only a few ofthe types of potential valence-restricted scaffolds that can beconstructed to meet this need.

Using the maleimide/succinimide moiety as a representative reactivegroup for this series of scaffolds, several potential compounds can besynthesized from commercially available starting materials. Illustratedin FIG. 6 is a sampling of these types of “point source scaffolds”. Oneskilled in the art will appreciate that the specific compounds describedcan be modified to produce point source scaffolds that utilize anynumber of alternative chemistries for conjugation or any size of “armlength” needed.

I. Valence Restricted Scaffolds Based on Cyclodextrins

An alternative type of scaffold can also be made that has the capabilityof being varied with respect to both the effective size and valence ofthe final construct. An example of this type of scaffold usingbeta-cyclodextrin as a template is illustrated below wherein the valencecan be controlled with precisely defined chemistry and the arm lengthusing various types of flexible spacers such as polyethylene glycol.

The cyclodextrins (CD) are oligosaccharides made up of glucose unitsthat are linked through α1→4 glycosidic bonds. In the resulting torusshaped molecule, the primary and secondary hydroxyls are positioned onopposing faces.

It is possible to selectively functionalize the primary hydroxyls in thepresence of the secondary hydroxyls. In addition, through the use of“linker groups” any combination of polyfunctionalized cyclodextrins canbe achieved (FIG. 7).

Perderivitization of α, β or γ CD provides the corresponding 6, 7 and 8valenced products. Each of these compounds can also bemono-functionalized. Treatment of β-CD, the most readily availablesubstrate, with a bifunctionalized protecting group will lead to the bisprotected product. This in turn can provide the 2 or 5 substitutedproducts. Accordingly, reaction with two linker groups leads to productswith valences of 3 and 4. Thus it is possible to attach between 1 and 8epitopes to CD by judicious use of protecting groups.

Using this type of chemistry, valence and “arm length” can be varied toproduce what can be considered as a radially disbursed array or“octopus-like” scaffold for ligand presentation. This type of array isoptimal for receptor/ligand interactions when the receptor population isrelatively free to move in the cell surface membrane. In addition, thechemistry of the “arms” can be varied to produce scaffolds withrelatively free range of motion to arms with progressively lessflexibility.

Disclosed below are some of the chemistries that can be employed to makethese types of constructs for use in suppressing an undesirable antigenspecific immune response.

β-Cyclodextrin 1, was transformed into its heptaamino derivative 2,using literature procedures (Boger et al, Helvetica Chimica Acta 1978,61:2910) (Scheme 1).

The extended arm product 3, was produced as follows heptaaminoβ-cyclodextrin (2) (3.0 g, 2.15 mmol) and triethylamine (2.4 mL, 17.2mmol) were dissolved in 50 mL DMF. EDC (3.78 g, 19.3 mmol) was addedfollowed by Boc-ε-aminocaproic acid (5.53 g, 19.3 mmol). The reactionmixture was stirred overnight, at which time 200 mL water was added anda precipitate formed. The solid product was filtered, washed with waterand dried under vacuum to yield 4.78 g (85%) of the Boc protected 3.Deprotection was effected as follows the product was dissolved in 50 mLof HCl saturated dioxane (4N) and stirred for 3 h. Evaporation followedto yield 3, 2.99 g (75%).

CS-0001 was produced from 3 as follows. The extended arm β-cyclodextrin(3) (500 mg, 0.23 mmol) was dissolved in 60 mL of 0.1M NH₄CO₃. GMBS(2.25 g, 8.05 mmol) was dissolved in 40 mL THF and added to the reactionmixture, which was stirred overnight. The mixture was evaporated andthen purified by RPHPLC to yield 251 mg (36%) of CS-0001.

An additional scaffold, CS-0002, was produced under the conditionsoutlined above; 3 was condensed with Boc-ε-aminocaproic acid followed byremoval of the Boc group. The resulting longer armed version of 3 wasthen reacted with GMBS to provide CS-0002 (Scheme 4).

The fluorescein specific construct CI-374 was produced from 3 asfollows. The extended arm β-cyclodextrin (3) (40 mg, 0.019 mmol) wasdissolved in 10 mL of 0.1M NaHCO₃. Fluorescein isothiocyanate (200 mg,0.52 mmol) was added and the resulting mixture was stirred overnight.The orange solution was then ultrafiltered through a YM3 membrane untilthe filtrate remained uncolored. The remaining orange retentate waspurified by RPHPLC to yield CI-374, 20 mg (16%).

The fourteen armed scaffold (6) was produced as follows. Heptaaminoβ-cyclodextrin (2) (500 mg, 0.36 mmol) was dissolved in 4 mL DMF.N^(α)-t-Boc-N^(ε)-t-Boc-L-lysine-N-hydroxysuccinimide ester (4.56 mg,10.1 mmol) was added followed by N-methylmorpholine (320 μL, 2.88 mmol).The reaction mixture was stirred overnight at which time 25 mL of waterwas added and a precipitate formed. The solid product was filtered,washed with water and dried. The resulting solid was dissolved in HClsaturated dioxane and stirred 3 h. Evaporation produced 575 mg (63%) of5.

This fourteen armed product-5 (400 mg, 0.16 mmol) was dissolved in 4 mLDMF. Boc-ε-aminoacaproic-N-hydroxysuccinimide ester (2.89 g, 8.85 mmol)was added followed by N-methylmorpholine (485 μL, 4.42 mmol). Theresulting mixture was stirred overnight, at which time 25 mL of waterwas added to effect precipitation of the product. The fourteen armedscaffold was isolated upon filtration, washed with water, and dried. Theresulting solid was immediately dissolved in HCl saturated dioxane andstired 3 h. Evaporation yielded 96 mg (60%) of 6.

These compounds have all exhibited satisfactory ¹H NMR, mass spectralanalysis and amino acid analysis.

Other arms can consist of polyethylene glycol units or some otherhydrophillic polymeric subunit. Spacers of this sort would permitexploration of distances between receptors. A heterobifunctional linkerwith amine and hydroxyl termini can be functionalized such that anactivating group can be fashioned at the hydroxyl terminus. This can inturn be displaced by the amines of compounds 2 or 3. Upon removal of theN-terminal protecting groups, a scaffold such as the ones previouslydescribed, containing longer spacer arms, will result.

Example 2

Synthesis and Analysis Procedures for Peptides used in ConjugatePreparation

a. Solid phase peptide synthesis

The peptides destined for incorporation into peptide-dextran conjugateswere generated by solid phase peptide synthesis using a standardstepwise elongation of the peptide chain. In brief, solid phase peptidesynthesis begins with N ^(α)-deprotection of the amino acid residueattached to the synthesis resin. This step is followed by neutralizationand washing of the deprotected amino acid-containing resin whichprepares it to receive (i.e. react with) the next amino acid, itselfactivated to facilitate the formation of the first peptide bond(—NH—CO—). A subsequent washing of the now (di)peptide-containing resinis then followed by the same series of events which are continued untilthe desired peptide has been produced. The finished peptide is thencleaved off of the resin under conditions which simultaneously removesome or all of the individual amino acid side-chain protecting groups.Specific protecting groups designed to be removed under differentconditions than that used for resin cleavage are frequently employed soas to render subsequent conjugation to backbone more controllable.

All reagents used in the studies described herein were obtained fromstandard commercial sources.

Solid phase peptide synthesis was carried out on either an AppliedBiosystems (ABI) 430A or Biosearch 9600 automated peptide synthesizerusing N ^(α)-tert-butyloxycarbonyl (N-t-BOC) protection. Trifunctionalamino acids other than Cys were protected with (protecting) groupscompatible with standard N-t-BOC solid phase peptide synthesis.N-t-BOC-L-Cys was S-protected with the p-methylbenzyl (Meb, HF labile),acetamidomethyl (Acm, HF stable) or nitropyridinesulfenyl (Npys, HFstable) group depending on the need for HF labile or HF stablesulfhydryl protection. The addition of a Cys residue to either the N- orC-terminus of a peptide destined for incorporation into a conjugateprovided the peptide with a nucleophilic moiety in the form of the Cyssulhydryl (—SH) group. Alternatively, other —SH containing residues (ex:cysteamine or homocysteine) can be substituted for cysteine in order toprovide an alternative conjugation moiety. The advantages of thesemodifications will be discussed below.

Finished peptidyl-resins were dried in vacuo and then placed in thereactors of a Biosearch HF cleavage apparatus or a PeninsulaLaboratories Type I HF apparatus. Peptides were cleaved from the resinusing standard HF procedures. After HF removal in vacuo, the resin waswashed well with diethyl ether and the peptide then extracted from theresin with trifluoracetic acid (with subsequent precipitation of thepeptide via the addition of diethyl ether) or with 10-30% aqueous aceticacid (with subsequent lyophilization).

Synthetic peptides purified by reverse phase high performance liquidchromatography (HPLC) were processed on a Waters Delta-Prep 3000preparative chromatography system (47 mm×30 cm Delta-Pak radialcompression cartidge containing 300 Angstrom, 15 μm C₁₈) equipped with avariable wavelength detector. Typically, peptides were eluted over a 40minute period with a linear acetonitrile gradient (0%-100%) containing aconstant concentration of trifluoroacetic acid (0.1% v/v). Thepurification was monitored at 215 nm and the homogeneity of purifiedmaterial was established by analytical HPLC on a Waters Delta-Pak C₁₈column (300 Angstrom, 15 μm C₁₈; column dimensions: 3.9 mm×30 cm) usingthe same gradient.

Amino acid analyses of synthetic peptides were obtained using the WatersPICO-TAG Chemistry (Bidlingmeyer, B. A., et. al., J. Chrom., 336, 93-104(1984)) which involves vapor-phase hydrolysis of peptides with constantboiling 6 M HCl, derivatization of the liberated amino acids withphenylisothiocyanate (PITC) and separation/quantitation of the resultingphenylthiocarbamyl (PTC)—amino acids by reverse phase chromatography ona Waters PICO-TAG C₁₈ column (5 μm C₁₈; column dimensions: 3.9 mm×15cm). The amino acid analysis of any purified peptide was consistent withits proposed sequence (accuracy of integrations: ±5%).

Example 3

Epitopes—Proteins

a. Epitope Mapping

Epitope mapping relates to the characterization of specific regions of aprotein that are being recognized by the immune system. It is unlikelythat peptide residues in the “core” of a globular protein are beingrecognized by the immune system at least as far as the development of ahumoral response is concerned. As a result, the surface map of a proteinwith respect to the different epitopes can be used to design andsynthesize peptides that can be incorporated into the desired array. Anexample of this type of epitope mapping is illustrated by theidentification of the histone antigen recognized by the NZB/NZW mouse.

In order to suppress the autoimmune response to histone H2B that occursin the (NZB×NZW) F, murine model of systemic lupus erythematosus (SLE),the antibody binding domain(s) of histone H2B had to be identified. Thisidentification process, referred to herein as “epitope mapping”,involves the synthesis of various overlapping peptide fragments whichare subsequently analyzed to establish regions of antigenicity. Clearly,the study of the entire H2B structure (125 amino acids) would require avery large amount of peptide synthesis. However, it is known fromstudies of SLE patients and mice with lupus-like disease that removal ofthe H2B N-terminal region with trypsin results in a loss of antigenicity(Portanova, J. P., et. al., J. Immunol. 38, 446-457, (1987)). Attentionwas, therefore, focussed on the synthesis of peptides derived from thisregion of histone H2B. The peptides synthesized together with theirrespective designations are shown in Table 2 below.

Sera obtained from (NZB×NZW) F, mice that were reactive to theN-terminal 30-mer (=Lupus 7′) of H2B were also found to bind strongly tothe peptide consisting of the first 15 amino acid residues (=Lupus 2′)but not to peptides consisting of more internal regions (i.e. Lupus 3′,4′ or 5′). Further characterization of the autoantigenic region of H2Binvolving peptides truncated from the N-terminus (=N-Ac-[Lupus 2′(6-15,5-15, 4-15, 3-15 and 2-15]—CONH₂) and from the C-terminus (=N-Ac-[Lupus2′ (2-12, 2-10 and 2-8]—COHN₂) resulted in it being possible to assignthe antigen recognized by (NZB×NZW) F, mice as being within H2B residues3-12.

TABLE 2 Histone H2B Peptides Synthesized for Epitope Mapping SEQ ID NO:1Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys-Ala-GlnLys-Lys-Asp-Gly-Lys-Lys-Arg-Lys-Ala-Tyr-Cys-CONH₂ = Lupus 7′. SEQ IDNO:2Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Cys-CONH₂ =Lupus 2′. SEQ ID NO:3N-Aceryl-Cys-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys-CONH₂= Lupus 3′. SEQ ID NO:4N-Aceryl-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys-Cys-CONH₂= Lupus 4′. SEQ ID NO:5N-Aceryl-Cys-Lys-Ala-Val-Thr-Lys-Ala-Gln-Lys-Lys-Asp-Gly-Lys-Lys-Arg-Lys-CONH₂= Lupus 5′. SEQ ID NO:6N-Aceryl-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-CONH₂ = N-Ac-[Lupus2′(6-15)]-CONH₂ SEQ ID NO:7N-Aceryl-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-CONH₂ = N-Ac-[Lupus2′(5-15)]-CONH₂ SEQ ID NO:8N-Aceryl-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-CONH₂ =N-Ac-[Lupus 2′ (4-15)]-CONH₂ SEQ ID NO:9N-Aceryl-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-CONH₂ =N-Ac-[Lupus 2′ (3-15)]-CONH₂ SEQ ID NO:10N-Aceryl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-CONH₂ =N-Ac-[Lupus 2′(2-15)]-CONH₂ SEQ ID NO:11N-Aceryl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-CONH₂ = N-Ac-[Lupus 2′(2-8)]-CONH₂SEQ ID NO:12 N-Aceryl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-CONH₂ =N-Ac-[Lupus 2′(2-10)]-CONH₂ SEQ ID NO:13N-Aceryl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-CONH₂ = N-Ac-[Lupus2′ (2-12)]-CONH₂ SEQ ID NO:14N-Aceryl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Glu-Cys(Acm)COOH,N-Ac-[Lupus 2′(2-13)]-Glu-Cys(Acm)-COOH SEQ ID NO:15N-Aceryl-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Glu-Cys(Acm)CONH₂,N-Ac-[Lupus 2′(2-13)]-Glu-Glu-Cys(Acm)-CONH₂

The peptide chosen to be incorporated into a suppressive conjugateobviously had to include enough immunological “information” to berecognized by the murine immune system but also had to address the netpositive (charge) character of residues 3-12. In order to accomplishthis, N-Ac-Glu², which is not required immunologically, was included aswere two non-histone C-terminal glutamic acid (Glu) residues. To the now“charge-balanced” peptide was added glycine, a residue also not requiredimmunologically but which provided space between those elements includedpurely for charge-balancing purposes (i.e. -Glu-Glu) and those requiredfor immunological recognition (i.e. residues 3-12). The final targetpeptide, designated N-Ac-[Lupus 2′(2-13)]-Glu-Glu-Cys-CONH₂ (see Table2), was then used for conjugation.

As mentioned above, highly cationic epitopes may need to be compensatedfor, particularly when they are arrayed in a multivalent way. In thiscase, such compensation was effected by adding additional anionic aminoacids to the defined epitope. As an alternative, an anionic scaffoldcould have been used. In either case, the desired outcome is to have anoverall charge neutral or slightly anionic construct so as to avoidnon-specific adherence of these compounds to anionic surfaces such ascell membranes.

The antigenic facade of the H2B histone protein consists of a singlecontinuous peptide sequence that was capable of accommodating the entirepopulation of antibodies generated by a population of mice. And, whileeach individual mouse recognized a discrete region within the entireepitope, the entire population of mice could be dealt with using asingle peptide ligand. This is unlikely to be the rule for otherproteins such as Ragweed antigen E where multiple discrete epitopes aremore likely to be encountered. Again, a certain amount ofmicroheterogeneity within a population with respect to a given epitopeis likely; no single epitope can be expected to predominate over all theothers for the entire population.

In view of the above, one of at least two alternatives can be employed.Either multiple ligands can be synthesized and presented either as amixture of arrays each with a specific ligand or an array of a mixtureof ligands (an artificial protein from an antigenic perspective) whereineach array contains a valence-restricted representation of the relevantligands. Another alternative is to produce valence-restricted arrays ofthe protein in question. Where these types of constructs are determinedto be the most appropriate means for manipulating the immune responsefor a specific antigenic protein, the following synthetic approaches canbe used.

b. Protein Oligomers as Oligovalent Heterogenous Epitope Arrays

An alternative to mapping the antigenic facade of a protein is toproduce oligomeric (valence restricted) arrays of the protein inquestion made up of either the protein crosslinked to itself or arrayedon a different type of scaffold. This type of construct is desirable ifthere are a large number of discrete epitopes that are being recognizedby the immune system or if some of the epitopes are formed bydiscontinuous conformationally constrained regions of the molecule. Twoof these types of constructs have been made and have been used to verifythat protein oligomers behave in accordance with the immunon paradigm.The preparation of these oligomers is described below.

(1) Polymerization of BSA and OVA

Conditions were established that allowed the polymerization of eitherBSA or of OVA to give polymers, in substantial yields, ranging fromdimers to very high polymers, all of which were water soluble and timestable. The properties of water solubility and time stability wereparticularly important because of the prolonged subsequent fractionationof the polymers on gel filtration columns, a procedure which producednarrow fractions of definable degrees of polymerization. BSA waspolymerized to itself through the use of a water soluble carbodiimide,1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a reagent which links afree carboxyl group on one molecule to a free amino group on aneighboring molecule through an amide bond. OVA was polymerized toitself by the use of glutaraldehyde, a reagent which links a free aminogroup on one molecule to a free group on a neighboring molecule.

(2) Fractionation Procedure

The protein monomers, oligomers and polymers, which were produced duringthe chemical cross-linking steps described above, were subjected tofractionation and repeated re-fractionation on a series of gelfiltration columns until they demonstrated narrow molecular weightdistributions, as measured by HPLC analysis. The molecular weight foreach fraction was then determined by the use of the Model E analyticalultracentrifuge under equilibrium conditions. During this prolongedseries of slow fractionation steps, molecules which were unstable to anyof the many steps involved in processing, handling, or storage werefractionated away from the samples, yielding a series of preparations,each of which contained a relatively narrow range of molecular sizes ofsubstantial time stability. These water soluble preparations were theninjected into mice intra-peritoneally, without the use of any adjuvant,in order to determine their relative immunogenicity. The level of immuneresponse was determined by measuring serum IgM or IgG antibody levelsagainst BSA or OVA by standard solid state ELISA technique.

Alternatively, the desired protein can be biotinylated in such a mannerthat only one biotin moiety is incorporated per protein monomer. Thiscan be accomplished by reacting a significant molar excess of theprotein monomer in dilute solution with a modified biotin moleculecapable of reacting with either free amines or carboxyl groups on theprotein. These conditions yield a predominance of “mono-functionalized”protein molecules with a minimum of multiply derivatized proteinmonomers. These biotinylated proteins can then be arrayed in arigorously tetravalent fashion with streptavidin with any polymericconstructs removed by size exclusion chromatography.

Similar “mono-functionalization” of a protein ligand can be achievedusing many different chemistries with the functionalized protein thenbeing arrayed on a valence restricted scaffold to achieve the sameendpoint. These valence restricted arrays can then be used to manipulatethe immune system in the desired fashion.

Finally, as previously mentioned, epitopes represented by well definedprotein domains or whole proteins can be incorporated into geneticallyengineered constructs having the desired valence for use as either aportion of or as a completely independent valence restricted array.

c. Mimotopes

Immunoglobulins and their related surface bound receptors arepredominantly concerned with the physical structure (shape,hydrophobicity or hydrophilicity, hydrogen bond donor or acceptorgroups, etc) and charge of the antigen in question. The specific“content” of the antigen with respect to peptide sequence, carbohydratecontent, etc. is only significant as it contributes to the “fit” of theligand to the receptor. As a result, there has been a considerableamount of work at a number of different laboratories directed atdeveloping methodologies that allow one to generate a multitude ofrandomly synthesized ligands that can be screened for their ability to“fit” a desired receptor. The relationship of a ligand identified inthis manner to the “natural” ligand to which the immune system'sresponse is directed is limited solely to their structural similarity.Such a ligand has been given the term “mimotope” to represent theability of this type of ligand to mimic a naturally occurring epitope.

One skilled in the art will appreciate that mimotopes can be modified toenhance their binding to the targeted receptor population using standardchemical modification techniques and substitutions. Mimotopes generatedby a random process may require modification prior to their beingconjugated to a scaffold to yield an agonist or antagonist array.

Example 4

Epitopes—Nucleic Acids

a. Size-fractionated Naturally Occurring DNA

Salmon testes DNA (Sigma) was digested with Aspergilus oryzae S1nuclease (Pharmacia) in order to eliminate “nicked” DNA. The product ofthis reaction was then subjected to partial digestion with bovinepancreatic Deoxyribonuclease I (BRL Gibco) in the presence of manganeseions. In the presence of manganese ions, bovine pancreatic DNase Icleaves both strands of a DNA duplex at approximately the same site toyield fragments of DNA that are blunt-ended or have protruding terminionly one or two nucleotides in length (Melgar and Goldthwaite, 1968).After cleavage with DNase I, the 5′ ends of the DNA retain the phosphategroups. The product of the DNase I reaction was then size-fractionatedon a 5 cm×92 cm Biogel A-1.5 m (fine mesh, Bio-Rad) column. The columnwas eluted with 150 mM NaCl, 50 mM Tris HCl pH 7.2, 1 mM EDTA. Fractionswere collected and aliquots of the fractions analyzed by polyacrylamidegel electrophoresis. Fractions that contained DNA of approximately 60 to120 nucleotides in length were pooled and the DNA recovered by ethanolprecipitation. The concentration of the DNA was determined by measuringthe OD₂₆₀, where 1 OD₂₆₀ equals 50 μg/ml double-stranded DNA. This DNAwas then modified for conjugation in the following manner.

Provided as the bis-5′-phosphate, the DNA (0.64 μmole) was convertedinto the bis-5′-(1-methyl)phosphorimidazolide with EDC (0.15 M) in1-methylimidazole buffer (0.1 M pH 6). Subsequent coupling to(S-3-nitro-2-pyridinesulfenyl)-cysteamine ((S-Npys)-Cmn, 0.2 M) thenafforded the bis-phosphoramidate; i.e. the conjugatable (followingdeprotection) form of the DNA. Removal of excess (S-Npys)-Cmn andisolation of the derivatized DNA was accomplished by repetitiveprecipitation from absolute EtOH.

b. Synthetic DNA

Alternative synthetic efforts designed to provide “conjugatable”synthetic DNA have focused on the 5′-length derivatization and eventualconjugation of synthetic DNA 40 nucleotides in length. DNA wassynthesized using β-cyanoethyl phosphoramidite chemistry on an AppliedBiosystems model 381A DNA synthesizer using the manufacturer's chemicalsand protocols. Some of the oligonucleotides were synthesized with anamino group at the 5′ end. This amino group was derived from acommercial DNA synthesis reagent, Aminolink 2 (Applied Biosystems). TheAminolink 2 reagent was used according to the manufacturer's recommendedprotocols. After removal from the solid support and deprotectionaccording to the manufacturer's protocols, the DNA was purified by gelfiltration on a 1.6 cm×16 cm column of Sephadex G-50 (fine mesh,Pharmacia). The column was eluted with 0.5 M NH₄OH. Fractions werecollected and those fractions containing the DNA were pooled andlyophilized. The DNA was resuspended in water and the concentrationdetermined by measuring the OD₂₆₀. For single-stranded DNA, 1 OD₂₆₀equals 40 μg/ml DNA. Derivatization is carried out with one of twoactivated S-containing amino acids:N-acetyl-S-Npys-L-cysteine-N-hydroxysuccinimide ester orN-(succinyl-N-hydroxysuccinimide ester)-S-Npys-cysteamine.

A reactive primary amino group can also be incorporated at the 5′-end ofthe synthetic DNA 40-mer via coupling of a modified nucleotide availablefrom Glen Research. This nucleotide, a modified thymidine, contains atrifluoroacetylated primary amine attached to the base moiety by a 10atom spacer group. As an alternative to the Aminolink approach, thismethod (of amine incorporation) has the advantage of verification ofincorporation of the nucleotide bearing the protected amino group (viastandard DNA calorimetric coupling assays). Chemical 5′-phosphorylationof synthetic DNA is also possible and yields DNA that is functionallyidentical to the size-fractionated DNA described above except that theresulting DNA is mono-functionalized. Preparing this type of syntheticDNA for conjugation, i.e. coupling of (S-Npys)-Cmn and subsequentdisulfide reduction, is accomplished as described in section a. above.Those skilled in the art will realize that phosphorothioate-containingDNA, and endo- and exonuclease resistant form of DNA, is equallyaccessible via solid phase DNA chemistry and can be modified by the sametechniques (described above) to generate conjugatable nucleic acid.

In all cases, the DNA intended for conjugation is derivatized with aprotected thiol-containing moiety that when deprotected and reacted withmaleimide containing scaffolds will conjugate to the scaffold in amanner analogous to the thiol-containing peptides described above. Inaddition, these modified DNA analogues now contain a residue that can beused to unambiguously confirm and quantitate covalent attachment of theDNA to the desired scaffold.

Example 5

Conjugates

A. Conjugate Synthesis

While the use of various types of sulfur based chemistries arespecifically described herein, these chemistries are but a smallsampling of the types that can be used for linking ligands andscaffolds. The types of chemistries that can be employed for providing“spacer arms” to reduce any steric interactions between the scaffold andligand or alternative conjugation chemistries for any particularapplication can be extrapolated from the foregoing disclosure. As longas the fundamental rules of valence and/or size are maintained and theligand can interact with the targeted receptor, any chemistry orgeometry of scaffold and ligand is acceptable. Some of the chemistriesthat have been employed to extend the immunon paradigm to the full rangeof immune responses are described below.

a. Small Molecular Weight Haptens

As mentioned below, the small molecular weight haptens chosen forinvestigation all included reactive groups that allowed for easycovalent attachment to the desired scaffolds. All of the conjugationswere done in aqueous solution and the unconjugated small molecularweight haptens removed by dialysis, ultrafiltration or size exclusionchromatography.

b. Peptides

The reaction between a cysteine (Cys)-containing peptide andGMB-dexamine (4) is but one example of the well-known tendency of thiol(—SH) nucleophiles to react with α, β-unsaturated carbonyl systems. Thisreaction is referred to as conjugate- or 1,4-addition and is used forthe covalent attachment of peptides to dextran (FIG. 8). Those skilledin the art will recognize that alternative conjugation chemistries canbe employed to accommodate any particular set of combinations ofbackbone or hapten. Alternative chemistries that take advantage of areactive thiol include reactions with haloalkanes or haloacetamides. Allsuch reactions involve a freshly-reduced buffered solution of a peptidebearing an N- or C-terminal Cys residue and freshly-preparedGMB-dexamine (4). C-Terminal Cys-containing peptides used in conjugationreactions were routinely purified to analytical purity by preparativeHPLC prior to conjugation. Although N-terminal Cys-containing peptidesmay not need to be purified prior to their conjugation and can, in fact,be used after only standard post HF cleavage extraction steps have beencarried out, these peptides were also purified prior to theirconjugation.

While the reaction of a Cys-containing peptide with GMB-dexamine (4) isan extremely rapid process (t_(½)<5 minutes for peptides conjugated atpH 5-7 in PBS, t_(½)=0.7 sec for the reaction of cysteine andN-ethylmaleimide at pH 7 in acetate buffer), there is one distinctcompeting reaction that occurs whenever a free sulfhydryl(Cys)-containing peptide and GMB-dexamine (4) are mixed together (inPBS, for example), i.e. the dimerization of the peptide to yield anunreactive disulfide. These two processes: conjugation and dimerizationoccurring simultaneously effect the ultimate conjugation yield and,hence, the density of conjugated peptide as well. The maleimidefunctional group has been observed to be extremely stable over the pHrange of the conjugation reaction (pH 5-7). Specifically, thesuccinimido-form of dexamine produced by hydrolysis of the maleimidedouble bond could not be detected after 2 days of exposure ofGMB-dexamine (4) to typical conjugation reaction conditions. Therefore,the hydrolysis of GMB-dexamine (4) does not appear to be a factor (i.e.a side reaction) influencing conjugation yield.

Cysteine (Cys)-containing peptides and GMB-dexamine (4) were prepared asdescribed above. Reductacryl resin (immobilized Cleland'sreagent=dithiothreitol (DTT)) was obtained from CALBIOCHEM Corp. with areduction capacity of ca. 0.5 meq/g. Phosphate-buffered saline (PBS)used in reduction ot Cys-containing peptides and in conjugationreactions was prepared as described above.

Purified peptides bearing N- or C-terminal Cys residues were dissolvedin PBS at concentrations up to 3 mg/mL. The peptide solution was thenadded to a 50-fold molar excess of reductacryl resin (DTT equivalents)in a glass reaction vessel equipped with a cinder glass bottom and anitrogen (gas) inlet. Reduction of the peptide was carried out for 30-45minutes at room temperature with gentle mixing of the reaction mixturepromoted by nitrogen bubbling. The fully-reduced Cys-containing peptidewas then added slowly to a freshly-prepared solution of GMB-dexamine (4)in PBS and the resulting conjugation reaction allowed to proceed for 2hours at room temperature (reaction pH 5-7). Since many conjugationreactions were carried out with sub-maximal levels of peptide, all wereroutinely quenched for 24 hours with a ten-fold molar excess ofmercaptoethanol (ME) relative to (dex)amine content. This precautionprevents the addition of unwanted peptides/proteins to the conjugateduring its in vivo lifetime or crosslinking of conjugate to each otheror to other macromolecules during purification, storage or use.Depending on the need for greater anionic character in the finalconjugate, mercaptoethanol can be replaced with either mercaptoaceticacid (MAA) or mercaptosuccinic acid (MSA) which introduce one or twoequivalents of negative charge, respectively, for each maleimide group.Purification of peptide-dextran conjugates:

Peptide-dextran conjugates present in the quenched reaction mixtureswere purified/isolated by either extensive dialysis or ultrafiltrationagainst PBS. Such treatment effectively removes mercaptoethanol or otherquenching reagent and unconjugated peptide from the finished conjugate.With regard to the latter type of reaction “contaminant”, the hypothesisthat noncovalently-associated peptide should not contributesignificantly to either the immunogenicity or to the immunosuppressivenature of a conjugate is reasonable when one considers what is expectedof a conjugate in the context of the Immunon model of immune response.In the absence of some type of covalent attachment “indicator”, however,noncovalently-associated peptide can result in significantoverestimation of the conjugate peptide substitution density, a quantityof great practical significance. An “unambiguous” indicator ofcovalently-bound conjugate peptide has been found(S-2-(2R,2S-succinyl)-L-Cys) and is discussed in greater detail below.Not addressed by either dialysis or ultrafiltration is the removal ofhigh molecular weight (i.e. significantly greater than that of thedesired conjugate) material from a finished peptide-dextran conjugate.Preliminary data indicate that, when present, high molecular weightmaterial is often only a very slight contaminant, i.e. the conjugatesare largely monomeric. (Because the observation of high molecular weightmaterial was made by laser light scattering analysis, it is not possibleto establish the exact extent of the contamination).

Quenched conjugation reaction mixtures were transferred in toto to12,000-14,000 mwco dialysis tubing and then dialyzed against PBSaccording to the following schedule: 24 hours against PBS containing ca.0.02% (w/v) NaN₃ (2 PBS changes), 24 hours against PBS (3 PBS changes)and then 24 hours against one-tenth strength (i.e. 15 mM NaCl and 1 mMphosphate, pH 7.3-7.4) PBS (3 PBS changes). Ultrafiltration was carriedout in an Amicon 8200 ultrafiltration vessel equipped with either a5,000 or 10,000 mwco filter as follows: The conjugation reaction mixturewas diluted to 200 mL total volume with PBS that contained ca. 0.02%(w/v) NaN₃ and then concentrated down to a volume≦30 mL at 55 psi(nitrogen pressure). The process was then repeated two times with PBSand two times with one-tenth strength PBS. Following the completion ofdialysis or ultrafiltration, purified peptide-dextran conjugate wasaliquotted into polypropylene-polyethylene vials, frozen andlyophilized. Finished conjugates were stored at −20° C. in lyophilizedform.

c. Nucleic Acids

As discussed above, the DNA used for conjugation is modified in such amanner that it will behave similarly to peptides when conjugated.Discussed below are some of the methods by which either the sizefractionated DNA or the synthetic DNA is attached to the desiredscaffold.

(1) Natural DNA

Deprotection of the DNA, i.e. removal of the S-Npys group, is carriedout via exposure to Reductacryl resin for 1 hour in 1 mM EDTA/1 MNaCl-containing 1-methylimidazole buffer (0.16 M, pH 6). The (now)thiol-containing DNA was then added directly into a solution of GMB-Dex(5 mg) to generate a DNA/Dex conjugate. Following quenching with L-Cysand reduction of the reaction volume by ultrafiltration, the conjugateis purified by preparative gel filtration on Sephacryl S-400 HR.Size-exclusion HPLC chromatography of the purified conjugate on a TSK5000 (Toso Haas) column indicated that complete removal of uncoupled DNAhad been achieved by the gel filtration step. The use of bis-thiolfunctionalized DNA made the production of oligomers (dextran moleculescross-linked with DNA) a likely possibility, and the presence ofoligomer in the concentrated conjugation reaction mixture was apparentduring preparative gel filtration. Although not completely resolved fromthe DNA/Dex “monomer” peak, this “contaminant” can be removed from thedesired conjugate if fractions were combined conservatively.

For valence calculation purposes, if it is assumed thatbis-functionalization of the DNA with reactive thiol (—SH) groupsresulted in the production of “looped” DNA structures on the dextransurface, then the quantitated amount of succinyl-cysteamine (Succ-Cmn)established by amino acid analysis will indicate one-half the amount ofcovalently-attached DNA. The conjugation chemistry described aboveappears to result in ca. 6-8 moles of “looped” DNA per mole ofDex_(7OK).

In the course of preparing the size-fractionated DNA-dextran conjugate,care was taken (in the form of excess Cys addition) to remove any traceof unreacted maleimide present on the GMB-dexamine. Furthermore, asmentioned above, the high molecular weight species present (i.e.oligomer) during preparative gel filtration was removed via conservativefraction combining. It was found, however, that upon standing at 4° C.the purified conjugate converted almost completely to the highermolecular weight oligomer. Since disulfide (—S—S—) dimers are likely toaccompany any reaction process involving bis-thiol-functionalizedmolecules and are the presumed source of the oligomer removed during thepreparative gel filtration, the most likely explanation for the observedmolecular weight shift is a continuation of the (presumed) disulfideformation process. Consistent with this hypothesis was the finding thatpurified conjugate responded to exogenously added DTT in the predictedmanner, i.e. essentially a complete regeneration of the desired lowermolecular weight conjugate.

Furthermore, size-fractionated DNA-dextran conjugate that had beenprepared months earlier and stored continuously at 4° C. responded toDTT but the rate of the reaction was much slower than that associatedwith freshly-prepared conjugate. These results are consistent with amore highly cross-linked (via disulfide bonds) preparation of conjugatedeveloping as a result of long storage. In order to prevent thereformation of disulfide bonds (inter-dextran), DTT reduction wasaccompanied by S-alkylation with excess maleimide.

Clearly, the tendency of this type of conjugate (or any type for thatmatter) to undergo a shift in molecular weight could significantlyconfound efforts to preare and administer an immunosuppressive agent.This example underscores the importance of completely characterizingconjugate material prior to its administration.

(2) Synthetic DNA

Following the isolation of modified (i.e. SH-containing) DNA,deprotection and conjugation is carried out as described above forsize-fractionated DNA. Single stranded DNA-containing (dextran)conjugate is then exposed to the complimentary DNA strand (also 40nucleotides in length) to afford the double stranded DNA-containing(dextran) conjugate. It is at this point that efforts to stabilize theresulting DNA duplex can be undertaken. Both the highly specificreagents: mitomycin C and the less specific psoralen can be used tocross-link the individual strands of the dextran-bound DNA duplex in anattempt to decrease the rate of exonuclease and (perhaps) endonucleasedigestion. Such a decrease would presumably result in a longer durationof action of the DNA-Dex conjugate and could, therefore, also result inlower doses of the conjugate being required for therapeuticintervention.

Alternatively, chemically modified DNA analogues such asphosphorothioates can be utilized. These nucleic acid analogues areknown to be resistent to endonuclease and exonuclease digestion.

B. Analysis

In all cases the resulting conjugates are subjected to rigorous analysiswith respect to both content and overall structure so as to assure thefinal product meets the criteria established for agonist or antagonistarrays as desired. Described below are representative analyticalprocedures for conjugates in general (haptens, peptides and nucleicacids) and the specific peptide containing conjugates used in theExamples set forth herein.

(a) Fractionation and Characterization of Haptenated Polymers

All of the procedures utilized for the preparation of hapten- orepitope-arrayed conjugates described above generated predominatelyhaptenated polymers of the desired molecular mass and degree of haptensubstitution. However, there was invariably present a substantial amountof material of higher molecular mass, which had been generated by asmall degree of unavoidable cross-linkage occurring between polymermolecules, due to side reactions. It was therefore necessary to purifythe haptenated or epitope-substituted polymer preparations further, byrepeated size fractionation on gel filtration chromatography columns asdescribed above, before they were homogeneous enough in molecular size(mass) for further chemical or physical characterization, and for use inimmunological studies.

Compositional Analysis of Haptenated Polymers

i) Dry Weight Analysis

The primary analytical reference standard for each type of polymericmaterial was a dry weight analysis for the actual amount of polymer masspresent in a given type of polymer preparation. Dry weight wasdetermined after the thorough vacuum drying of polymer samples andappropriate dialysate samples.

ii) Spectral Analysis

The haptens used in these studies were chosen, in part, so that they hadidentifiable spectral absorption bands at wavelengths in the visible ornear ultraviolet regions. The amount of hapten chemically coupled topolymer molecules in a preparation was usually determined from thecomparison of the optical absorption due to the hapten groups and thetotal mass of the polymer preparation, as determined by dry weightanalysis or refractive index increment analysis.

iii) Chemical Analysis

It was sometimes possible to measure the quantity of a certain type ofchemical group present on polymer molecules by direct chemical analysis.Amino groups were often measured by reaction withtrinitrobenzenesulfonic acid, yielding a colored product which couldthen be measured by spectrophotometric analysis.

Carbohydrate could be determined by reaction with sulfuric acid andphenol, to give colored products having measurable optical absorption.The benzyl penicillin hapten could be measured by reaction with amercurial compound to give colored products. Peptide and/or proteincontaining conjugates can be characterized by amino acid analysis (seebelow).

iv) Refractive Increment Analysis

By the careful calibration of the refractive index increment due to thepolymer against the dry weight measurement for each type of polymermolecule, it was possible to substitute refractive index incrementmeasurement for dry weight measurement. This procedure has greatlyincreased the accuracy of measurement of polymer mass by permittingsensitive and accurate measurement of the mass of the polymericmaterials during HPLC measurements on size exclusion columns.

v) Titration Analysis

Chemical groups with ionization constants near the neutral range, suchas carboxyl, amino and phenolic groups, could be measured directly bymeans of acid-base titration. This procedure was especially importantfor the measurement of carboxyl group content, since the carboxyl groupis very difficult to measure by spectrophotometric means in aqueoussolutions of polymers. Such measurement of ionizable chemical groups isespecially important in determining the net electrical charge on largepolymer molecules, a parameter which affects the interaction of thepolymer molecules with electrically charged cell surfaces.

(b) Determinations of the Molecular Mass and Size of Haptenated PolymerPreparations

i) Size Exclusion Chromatography (SEC)

Use of SEC methods permit the convenient determination of relativemolecular mass by comparison of the chromatographic column retentiontimes of unknown samples and homogeneous standard samples using standardHPLC techniques. The standardization polymer materials have to berelatively homogeneous and independently calibrated for molecular massby some absolute experimental procedure, such as equilibriumultracentrifugation or low angle laser light scattering. Because the SECmethod is very sensitive to any physical interactions between the columnsupport and the polymer molecule, the column retention times must becalibrated for each and every type of haptenated polymer molecule. Suchcalibration is sensitive to the physical and chemical nature of thepolymer molecule, the chemical nature and number of haptens, the netelectrical charge on the molecule, etc.

ii) Equilibrium Analytical Ultracentrifugation

When appropriately combined with another experimentally measurablequantity—the partial specific volume, short column equilibriumanalytical ultracentrifuge measurements yielded the absoluteweight-average molecular weight of a substantially homogeneous polymerpreparation. The experimental method requires a series of measurementsat several polymer concentrations followed by the extrapolation of theresults to zero polymer concentration. Equilibrium measurements are madeat different centrifuge speeds of rotation, in order to demonstrate therelative independence of the extrapolated molecular weight on rotorspeed. FIG. 9 illustrates for a particular fluorescein-dextran samplethe determination of molecular weight as determined at two differentrotor speeds, (14,000 and 16,000 RPM) and by extrapolation to zeropolymer concentration. In this case the weight average molecular weightof the haptenated polymer was approximately 68 kDa.

iii) Low Angle Laser Light Scattering

When appropriately combined with an experimentally determined refractiveindex increment, low angle light scattering methods yield a value forthe absolute weight average molecular weight of a polymer preparation.For large molecules, the method requires measurements at a number ofconcentrations and angles, followed by extrapolation both to zeropolymer concentration and to zero angle of measurement.

If combined with the separation of molecules of different molecularsizes using SEC, this light scattering method yields a dependabledetermination both of the molecular mass and physical size distributionsin a very small quantity of polymer preparation. Measurements of thistype were routinely carried out using a high pressure liquidchromatography apparatus, Hewlett-Packard HP1090M, and a low angle laserlight scattering device, Wyatt Technology Dawn. Size exclusion columnswere Toyo Soda TSK GMPW gel columns or Pharmacia Superose 6 or 12columns or combinations thereof, appropriately chosen to separate themolecular sizes present in the particular sample. When low molecularweight samples were inadequately separated from salt a column ofSephadex G15 was added to increase resolution. When polymers weresubstituted with high amounts of haptens having appreciable hydrophobiccharacter, such as dinitrophenyl or fluorescein, there was significantinteraction between the hapten and the column material, causinginterference with the size exclusion based separation. When this effectoccurred, it was minimized by using 20% acetonitrile in the columnbuffer.

Examples of the results obtained with peptide-dextran conjugatesanalyzed by the low angle laser light scattering method are shown inTable 3. The data show the results obtained in different runs, usingdifferent combinations of size exclusion columns. Aside from a fewinstances where obvious technical problems were present, the data showsubstantial consistency and are in general agreement with expectationsfor the particular samples.

TABLE 3 Sample Columns mn mw mw/mn  10K Dexamine SUP-6 + 12 12.8K  13.5K 1.05 COR-3255 GMPW + G-15  13.5K  16.1K 1.19 G-15 + GMPW  10.0K  11.0K1.10  40K Dexamine SUP-6 + 12 48.0K  51.2K  1.07 COR-3254 GMPW + G-15 64.4K  74.6K 1.16 G-15 + GMPW  45.5K  48.2K 1.06  70K Dexamine SUP-6 +12 66.1K  88.1K  1.33 COR-3255 GMPW + 2.5K  68.7K  82.6K 1.20 G-15 +GMPW  62.6K  72.8K 1.16 500K Dexamine SUP-6 + 12 307K 440K  1.43COR-3256 GMPW + 2.5K 403K 460K 1.14 G-15 + GMPW 317K 441K 1.39 A GMPW +2.5K  23.4K  30.8K 1.32 COR-3257 G-15 + GMPW  22.8K  25.7K 1.13 BSUP-6 + 12 99.1K 223K  2.25 COR-3258 GMPW + 2.5K  96.1K 147K 1.53 G-15 +GMPW  99.1K 141K 1.42 C SUP-6 + 12 685K  1.38M  2.01 COR-3259 GMPW +2.5K 397K  1.04M 2.62 G-15 + GMPW 586K  1.08M 1.84 D SUP-6 + 12 48.2K 55.9K  1.16 COR-3260 GMPW + 2.5K  34.1K  40.1K 1.18 G-15 + GMPW  28.4K 31.0K 1.09 E SUP-6 + 12 676K  1.45M  2.14 COR-3261 GMPW + 2.5K 408K 1.05M 2.57 G-15 + GMPW 557K  1.02M 1.83 F SUP-6 + 12 113K 130K  1.15COR-3262 GMPW + 2.5k  78.4K  92.5K 1.18 G-15 + GMPW  83.9K  93.2K 1.11

(c) Analysis of Peptide-dextran Conjugates

The importance of analyzing peptide-dextran conjugates relatesultimately to the expectation that different immunological behavior willbe elicited by conjugates having different peptide substitutiondensities. Amino acid analysis via the Waters PICO-TAG chemistryfollowing the complete acid (HCl) hydrolysis of a peptide-dextranconjugate has been found to be a very effective method for measuringboth (conjugate) peptide and carbohydrate content. Although acidhydrolysis does not permit recovery of the peptide or carbohydrateportions as intact entities, such recovery is not necessary for theevaluation of conjugate peptide substitution density. That is, simply byrecovering and quantitating the amino acids derived from the conjugatedpeptide and GMB-dexamine it is possible to assess the moles of boundpeptide and the moles of recovered dexamine, respectively.

The typical products derived from the acid hydrolysis of apeptide-dextran conjugate are shown in FIG. 10. The three components ofinterest are: gamma-aminobutyric acid (GABA); which indicates themaximum amount of peptide that can be conjugated covalently and whichalso provides a direct measure of the amount of recovered dexaminebackbone, S-2-(2R,2S-succinyl)-L-Cys; which distinguishescovalently-bound from noncovalently-bound conjugate peptide (becauseonly covalently-bound peptide is S-succinylated) and the amino acidsderived from the conjugated peptide. The phenylthiocarbamyl(PTC)-derivative of S-2-(2R,2S-succinyl)-L-Cys, having a retention timeof 1.33 minutes on the PICO-TAG HPLC column, is well separated from anyother PTC-derivative. The PTC-derivative of gamma-aminobutyric acid(PTC-GABA), however, coelutes with that of arginine (Arg). While this isinconvenient when Arg-containing peptide-dextran conjugates are beinganalyzed, difference analysis (i.e. pmoles @ GABA=total pmoles inpeak—pmoles @ Arg) can be used to measure GABA recoveries when integralpmole values of other conjugate peptide amino acids are known. Anexample of the type of analytical data obtained from PICO-TAG conjugateanalysis is shown in FIG. 11 which displays the HPLC chromatogram of thePTC-amino acids derived from a (lupus) histone peptide-dextranconjugate.

The importance of attending to the problem of differentiating betweencovalently—vs. noncovalently-bound conjugate peptide as well as topotential losses of conjugate that may occur throughout the productionprocess is exemplified in FIG. 12. The ultimate goal of the conjugateanalysis process is to measure as accurately as possible the number ofpeptide molecules bound per average molecule of dextran. Although theequation which yields this information is simple enough (FIG. 12),several variables effect the numerator and the denominator of thisequation. Quantitation of the PTC-derivatives of S-2-(2R,2S-succinyl)-L-Cys and GABA, however, significantly increases theaccuracy of conjugate peptide substitution density measurements.

Although the importance of the PTC-derivative ofS-2-(2R,2S-succinyl)-L-Cys cannot be overemphasized, other sulfur(S)-containing amino acids have been found which can also provide anunambiguous assessment of covalently-attached conjugate peptide.Specifically, the PTC-derivatives of S-2-(2R,2S-succinyl)-cysteamine(retention time=3.38 minutes) and S-2-(2R,2S-succinyl)-DL-homoCys(retention time=1.50 minutes) (see FIG. 13), derived from thecorresponding cysteamine- or homocys-containing peptide/GMB-dexamineconjugate, can substitute effectively forPTC-(S-2-(2R,2S-succinyl)-L-Cys. The ability to generate and quantitatethree different sulfur (S)-containing “marker” amino acids can be ofvalue when different peptides are conjugated to the same sample ofGMB-dexamine (4).

Provided that purification of a peptide-dextran conjugate is complete,non-sulfur containing amino acids may function in a similar context(i.e. as marker amino acids). In particular, amino acids not normallyfound in the biologically relevant portion of peptides to be conjugatedsuch as δ-aminovaleric acid (δ-AVA), ε-aminocaproic acid (ε-ACA),β-alanine (β-Ala), norleucine (Nle), norvaline (Nva) and α-aminobutyricacid (α-ABA), can be regarded as “markers” that specify the amount of aparticular covalently-attached peptide. When located penultimately to areactive Cys, cysteamine or DL-homoCys residue, these amino acids mayalso be thought of as “spacer” elements that provide distance betweenthe chemically reactive (i.e. S-containing) and biologically relevantportions of a peptide destined for incorporation into a peptide-dextranconjugate.

It is advantageous to purify C-terminal Cys-containing peptidescompletely by reverse phase HPLC before using this type of peptide in aconjugation reaction. The reason for the rigorous purification of thesepeptides prior to their use relates directly to the manner in whichpeptides are synthesized by solid phase techniques, i.e. from theC-terminus. All deletion peptides or failure sequences associated with aC-terminal Cys-containing peptide will also possess the reactive (i.e.—SH-containing) Cys residue. As a result, these unwanted peptides couldbe expected to conjugate to GMB-dexamine (4) in the same way that thecompleted or desired peptide would. Such a “series” of reactions wouldlead to a conjugate that is really a composite of all possible deletionpeptides derived from the full-length peptide of interest. In thisregard, attention is directed to data shown in Tables 4 and 5.

TABLE 4 SAMPLE NAME: CI-0060/1420K Dex (1:1) Pure Peptide AA Conc. Mole% Moles Int. ASP 0.00 0.000 0.00 0 GLU 46.50 0.071 1.06* 1 SER 84.640.129 1.94 2 GLY 45.65 0.070 1.04* 1 HIS 0.00 0.000 0.00 0 ARG 0.000.000 0.00 0 THR 0.00 0.000 0.00 0 ALA 140.33 0.214 3.21 3 PRO 172.250.263 3.94 4 TYR 0.00 0.000 0.00 0 VAL 0.00 0.000 0.00 0 MET 0.00 0.0000.00 0 ILE 0.00 0.000 0.00 0 LEU 0.00 0.000 0.00 0 PHE 0.00 0.000 0.00 0LYS 165.89 0.253 3.80 4 CI-0060 = Lupus 2′:Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Cys-CONH₂SEQ ID NO: 2 378 molecules peptide/molecules Dexamine (Max: ˜1024) ABARecovery: 68% *Glu/Gly = 1.02

TABLE 5 SAMPLE NAME: CI-0060/1420K Dex (1:1) Impure Peptide AA Conc.Mole % Moles Int. ASP 0.00 0.000 0.00 0 GLU 30.39 0.062 0.93* 1 SER64.77 0.133 1.99 2 GLY 41.34 0.086 1.29* 1 HIS 0.00 0.000 0.00 0 ARG0.00 0.000 0.00 0 THR 0.00 0.000 0.00 0 ALA 98.69 0.202 3.03 3 PRO118.85 0.243 3.65 4 TYR 0.00 0.000 0.00 0 VAL 0.00 0.000 0.00 0 MET 0.000.000 0.00 0 ILE 0.00 0.000 0.00 0 LEU 0.00 0.000 0.00 0 PHE 0.00 0.0000.00 0 LYS 133.82 0.274 4.11 4 CI-0060 = Lupus 2′:Pro-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Ser-Lys-Cys-CONH₂SEQ ID NO: 2 247 molecules peptide/molecule Dexamine (Max: ˜1024) ABARecovery: 74% *Glu/Gly = 0.72

The pmole ratio of an amino acid near the N-terminus (Glu) to an aminoacid near the C-terminus (Gly) should be equal to one for the lupuspeptide (CI-0060)/1420K dextran conjugate prepared for this experiment.The fact that this ratio is significantly less than one in the casewhere the impure C-terminal Cys-containing peptide was conjugated (Table5) is consistent with a mixture of peptides actually participating inthe conjugation process.

Conversely, even though an N-terminal Cys-containing peptide is alsocontaminated with deletion peptides after it is cleaved from the resin,none of these peptides should possess the reactive Cys residue. That is,only the peptide of interest (which is the finished peptide) should beable to undergo a conjugation reaction. That this is indeed the case isdemonstrated by the data in Tables 6 and 7. Again, the pmole ratio of anamino acid near the N-terminus (epsilon-aminocaproic acid=ε-ACA) to anamino acid near the C-terminus (Pro) should be approximately equal toone for the peptide (CI-0134)/65K dextran conjugate prepared for thisexperiment.

TABLE 6 SAMPLE NAME: CI-0134/65K Dex (1:1) Pure Peptide AA Conc. Mole %Moles Int. ASP 27.58 0.091 2.09 2 GLU 26.72 0.088 2.03 2 SER 12.15 0.0400.92 1 GLY 79.88 0.263 6.06 6 HIS 0.00 0.000 0.00 0 ARG 27.00 0.089 2.052 THR 0.00 0.000 0.00 0 ALA 27.12 0.089 2.06 2 PRO 13.83 0.046 1.05* 1TYR 11.40 0.038 0.86 1 VAL 38.42 0.127 2.91 3 MET 0.00 0.000 0.00 0 ILE0.00 0.000 0.00 0 LEU 13.71 0.045 1.04 1 PHE 13.97 0.046 1.06 1 LYS 0.000.000 0.00 0 ACA 11.43 0.038 0.87* 1 CI-0134 =Ac-Cys-(ε-ACA)-Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Val-(d)Tyr-CO₂HSEQ ID NO: 16 9 molecules peptide/molecule Dexamine (Max: ˜54)*ε-ACA/Pro = 0.83

TABLE 7 SAMPLE NAME: CI-0134/65K Dex (1:1) Impure Peptide AA Conc. Mole% Moles Int. ASP 23.28 0.093 2.14 2 GLU 22.42 0.089 2.06 2 SER 11.000.044 1.01 1 GLY 66.75 0.266 6.13 6 HIS 0.00 0.000 0.00 0 ARG 22.000.088 2.02 2 THR 0.00 0.000 0.00 0 ALA 22.57 0.090 2.07 2 PRO 10.850.043 1.00* 1 TYR 8.13 0.032 0.75 1 VAL 31.13 0.124 2.86 3 MET 0.000.000 0.00 0 ILE 0.00 0.000 0.00 0 LEU 11.44 0.046 1.05 1 PHE 11.580.046 1.06 1 LYS 0.00 0.000 0.00 0 ACA 9.36 0.037 0.86* 1 CI-0134 =Ac-Cys-(ε-ACA)-Ala-Asp-Ser-GlyGlu-Gly-Asp-Phe-Leu-Ala-Glu-Gly-Gly-Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Val-(d)Tyr-CO₂HSEQ ID NO: 16 8 molecules peptide/molecule Dexamine (Max ˜54) *ε-ACA/Pro= 0.86

The fact that the ratio is essentially the same when either a pure or animpure N-terminal Cys-containing peptide is used to generate theconjugate suggests that the impure peptide undergoes a “purification ofsorts” as a result of its participation in the conjugation process. Inorder to obtain the highest possible purity of conjugate, all peptidesare purified to analytical purity prior to conjugation.

All reagents used in the studies described herein were obtained fromstandard commercial sources.

Purified peptide-dextran conjugates were routinely dissolved inHPLC-grade water at a concentration of ca. 1 mg/mL. An appropriatealiquot was removed, dried in vacuo and then subjected to the WatersPICO-TAG chemistry (see above) for amino acid analysis.

Proton ('H) nuclear magnetic resonance (NMR) spectra were recorded on aVarian Associates Gemini-300 300 MHz spectrometer in deuterateddimethylsulfoxide (DMSO). Chemical shift values are relative to addedtetramethylsilane (TMS) as the internal standard. All peaks areexpressed as ppm downfield from TMS.

Thin layer chromatography (TLC) was performed on Merck (#5715) Silicagel plates. Products were visualized by Cl₂/starch-KI stain and/orninhydrin reactivity .

N ^(α)-Acetyl-S-3-(3R,3S-succinimido)-L-Cys was prepared as follows: Toa stirred solution of N-Ac-L-Cys (0.082 g, 0.50 mmole) in 10 mL of H₂Owas added NMM (0.101 g, 1 mmole) and maleimide (0.0485 g, 0.50 mmole).The reaction mixture was stirred overnight at room temperature and thentransferred in toto to a 25 mm×22 cm Dowex AG50W-X₄ column. The columnwas eluted with H₂O and fractions (25×8 mL) were collected and analyzedby TLC. The desired Cys derivative was found in fractions 8-14. Thesefractions were combined and lyophilized to give a fluffy, white solid.Yield: 0.074 g (0.285 mmole, 57%) mp 76-81° C. TLC (n-butanol: aceticacid: H₂O (4:1:1)): R_(I)=0.43. NMR: δ1.86 (s, 3H), δ2.43 (m, 1H), δ2.90(dd, 1H), δ3.00-3.30 (m, 2H), δ3.95 (m, 1H), δ4.23 (m, 1H), δ8.31 (d,J=7.8 Hz)+δ8.34 J=7.8 Hz)=1H, δ11.39 (s, 1H).

Hydrolysis of N ^(α)-acetyl-S-3-(3R,3S-succinimido)-L-Cys with (vaporphase) 6 M HCl in preparation for PICO-TAG analysis gave the amino acidstandard: S-2-(2R,2S-succinyl)-L-Cys in quantitative yield.

A dialytic or ultrafiltrative purification has proven very satisfactoryin the initial stages of the preparation of peptide-dextran conjugates.Certain applications may, however, require conjugate preparations thatare completely devoid of high or low molecular weight impurities.Molecular exclusion chromatography of dextran samples on Superose 12 orSuperose 6 can be very effective as a means of sample purification. Acommercially available analytical Superose 12 column (Pharmacia)attached to an in-house fast protein liquid chromatography (FPLC) systemwill separate fluoresceinated dextran (Fl-Dex) samples reasonably well.A preparative Superose 12 column (separation range: 1,000-3×10⁵ g/mole)could be used to purify large-scale reaction mixtures of peptide-dextranconjugates. Separation results obtained from the preparative Superose 12column using Fl-Dex standards suggest that this type of chromatographymay be useful both as a means of conjugate purification and (expensive)peptide recovery.

Using these techniques, the conjugates used in the anti-histone,anti-OVA and anti-EALA studies described herein were characterized asfollows:

Histone Peptide Conjugates:

1) 65K Dextran 0.2:1 molar reaction→8 mole peptide/mole dex. (=CI-0125)(=CI-0084/Dex_(65K)(0.2:1)).

2) 65K Dextran 2:1 molar reaction→35 mole peptide/mole dex. (=CI-0126)(=CI-0084/Dex_(65K) (2:1)).

CI-0034=N-Ac-Glu-Pro-Ala-Lys-Ser-Ala-Pro-Ala-Pro-Lys-Lys-Gly-Glu-Glu-Cys-CONH₂SEQ ID NO: 17

Ova Peptide Conjugates:

Analysis of the purified suppressive conjugates gave the followingresults:

1) 40K Dextran 0.3:1 molar reaction→2 mole peptide/mole dex. (=CI-0252)(=CI-0159/Dex_(40K) (0.3:1)).

2) 40K Dextran 1:1 molar reaction→10 mole peptide/mole dex. (=CI-0253)(=CI-0159/Dex_(40K) (1:1)).

CI-0159=N-Ac-Cys-(ε-ACA)-Glu-Ala-His³³¹-Ala-Glu-Ile-Asn-Glu-Ala-Gly-Arg³³⁹-CONH₂SEQ ID NO: 18.

EALA Peptide Conjugates

1) 84K Dextran 1:1 molar reaction→57 mole peptide/mole dex. (=CI-0218)(=CI-0010/Dex_(84K) (1:1)).

CC-0010=Cys-Gly-Ala-Gly-(Glu-Ala-Leu-Ala)₆-Gly-Ala-Gly-Arg-Gly-Asp-Ser-Pro-Ala-CONH₂SEQ ID NO: 19.

Characterization of DNA-dextran conjugates by amino acid analysisinvolves the same type of manipulations as those that accompany theanalysis of peptide-dextran conjugates. As a result of acid hydrolysis(6 M HCl, 110° C., 22-24 hours), the conjugate is degraded to yield: DNAnucleotide hydrolysis peaks, succinyl-Cys (Succ-Cys) onsuccinyl-cysteamine (Succ-Cmn), and -aminobutyric acid (GABA, see FIG.14). (The carbohydrate derived from dextran is not recovered in aquantitatable form). As is true of peptide-dextran conjugates, theimportance of the liberated S-containing amino acid (Succ-Cys orSucc-Cmn) cannot be overemphasized because it ultimately providesunambiguous assessment of the level of covalent attachment of the DNA tothe dextran polymer. This measure of conjugation combined with thatprovided by quantitation of the DNA nucleotide hydrolysis products (seeFIG. 15) and direct absorbance measurement at 260 nm will result inthree independently generated assessments of the amount of conjugatedDNA. Taken together with the recovered GABA value, which is used toquantitate dextran recovery, conjugate DNA substitution density can beestablished.

Example 6

Linear polyacrylamide substituted with Dnp hapten groups was prepared asdescribed above. Thus, linear polyacrylamide (Gelamide 250-AmericanCyanamid) with average molecular weight 5×10⁶ was substituted withethylene diamine in a manner analogous to that previously used forpolyacrylamide beads (Inman et al, Biochemistry 8, 4074-4082 (1969)).Dnp derivatives were obtained by shaking the ethylene diaminesubstituted derivatives with excess fluorodinitrobenzene followed byextensive dialysis. The degree of substitution was determined frommeasurement of dry weight and optical absorbance at 360 nm. Preparationswere labeled with ¹²⁵I substitution levels of approximately one per 2500monomer units were obtained, corresponding to less than one ¹²⁵I permolecule labeled.

Dnp-substituted polymers were fractionated by gel filtration through 1 mlong columns of Bio-Gel A-0.5 M agarose beads. These original fractionswere further fractionated three more times to obtain relativelyhomogeneous preparations, as determined by sedimentation equilibriummeasurement in the analytical ultracentrifuge.

Two Dnp-substituted polymer preparations were obtained having thefollowing characteristics:

Polymer B Polymer D Molecular weight, × 10⁻⁵   0.8   1.8 Acrylamidemonomer subunits/ 1050 2350 molecule Extended length of polymer 25006000 chain, A Acrylamide monomer subunits/  42  36 Dnp Average distancebetween Dnp  105  90 groups, A Total Dnp groups/molecule  25  66“Effective” Dnp groups/molecule   8-12  22-33

Polymer B was not immunogenic while Polymer D was (see Table 1, 1976paper noted above).

Polymers B and D were subjected to further column fractionation onSepharose Cl-4B. Two preparations (N and S) were separated for furthertesting. Preparation N was a central subfraction of polymer B andpreparation S was a central subfraction of polymer D. Measurement ofpartial specific volume (0.690 ml/g) and extrapolation of sedimentationequilibrium molecular weight to zero concentration gave values of 60,000for N and 130,000 for S. These values together with dry weight andabsorbance at 360 nm show N to contain 19 Dnp groups per molecule [7-9“effective” or appropriately spaced] whereas S contains 43 Dnp groupsper molecule (14-21 “effective”). Polymers N and B had almost identical“epitope densities” or degrees of substitution by hapten per molecularsize unit.

Antibody Response. Polymer preparations were injected intraperitoneallyin BALB/c mice in 0.5 ml of isotonic saline. After 6 days, blood wascollected by bleeding from the tail, and the serum was stored at −30° C.until analysis. The concentration in serum of IgM antibody against Dnpwas determined by a solid-phase binding assay. Surfaces covalentlycoated with Dnp-substituted gelatin served to bind the anti-Dnp mouseantibody, whose presence was then measured by a second incubation withI¹²⁵-labeled rabbit antibody against mouse IgM antibody.

In Vitro Culture and Assay. Mice were killed by cervical dislocation,and their spleens were minced in RPMI-1640 medium and pressed through astainless steel mesh (60×60 mesh; 0.019-cm diameter). Cellular debriswas allowed to settle, and the supernatant containing a dispersed-cellsuspension was decanted, freed of erythrocytes by osmotic shock, andwashed. Suspensions of nucleated spleen cells were then incubated withor without appropriate polymer in 60×15 mm tissue culture dishescontaining 5×10⁷ viable cells in a final volume of 7.5 ml. Theincubation was carried out in 5% CO₂/95% water-saturated air at 37.0° C.The incubation medium consisted of RPMI 1640 medium enriched with 5%(vol/vol) heat-inactivated fetal calf serum, 2% (vol/vol)heat-inactivated horse serum, 4 mM glutamine, 100 units of penicillinand 100 μg of streptomycin per ml, and 50 μM 2-mercaptoethanol.

After 3 days of incubation, cells were harvested and washed. Assay fordirect (IgM) anti-Dnp plaque-forming cells was performed.

The immunological response in BALB/c mice 6 days after injection ofvarious doses of immunogenic polymer preparation S, as measured by theconcentration of serum IgM molecules reactive toward Dnp groups, isshown in FIG. 16. The mice in this experiment came in a single shipmentof uniform age from the supplier and were divided into groups of 10.Members of each group were injected with the same dose, and all groupswere handled as uniformly as possible. The solid curve in FIG. 16 is thetheoretical response curve expected from Eq 1 $\begin{matrix}{r = {\frac{D_{s}}{D_{s}^{\prime}}\quad\left\lbrack \frac{{\left( {q - 1} \right)D_{s}^{\max}} + D_{s}^{\prime} + D_{N}^{\prime}}{{\left( {q - 1} \right)D_{s}^{\max}} + D_{s} + D_{N}} \right\rbrack}^{q}} & \lbrack 1\rbrack\end{matrix}$

as visually fitted to the experimentally determined points by adjustmentof the numerical value of D_(S) ^(max) to 0.3 μg. It has been shown byDintzis et al (see Proc. Natl. Acad. Sci. USA 79:395 (1982)) that ifdoses D_(S) of immunogen and D_(N) of nonimmunogen are injected into oneanimal and doses D′_(S) and D′_(N) are injected into a second animal,then the ratio r of immune response in the first animal relative to thatin the second animal should be given by Eq. 1 where D_(S) ^(max)corresponds to the dose of immunogen giving maximum response in ananimal—i.e., the peak of the dose-response curve.

In view of the simplicity of the assumptions involved in the derivationof Eq. 1 and the known variability of response of individual mice, theagreement between theory and experiment is surprisingly good. However,when the experiment was repeated by using different groups of micesupplied by the same breeder,the variability of biological responses inwhole animals became more evident.

FIG. 17 compares the dose-response curves of three separate shipments ofBALB/c mice and illustrates both group-dependent variability of responseof individual mice at each dose and some change of shape of thedose-response curve from group to group. The variable immunologicalresponse given by different groups of mice is a well-known phenomenon,having been observed both in studies using whole animals and in thoseusing cell cultures. It probably is dependent on factors in the previoushistory and handling of the animals, such as exposure to bacteria,viruses, and parasites, which might influence the “antigenic naivete” ofthe animals, as well as exposure to environmental shocks such as heatand cold during shipment.

By comparing the observed dose-response curves shown in FIGS. 16 and 17with the theoretical curve shown in FIG. 16, it is clear that althoughthe agreement between curves is good, the observed responses are quitevariable from one batch of mice to another and, in general, show a widerdose-response curve than expected from the simple model that generatedthe curve shown in FIG. 16.

The wider experimental curve may be explained in the following way:

The theoretical curve in FIG. 16 is based on the assumption that allcells responding to the immunogen have receptor molecules with the samebinding constant for Dnp groups. This assumption of complete homogeneityis unlikely to be true. If cells that bind immunogen and respond to ithave protein receptors with differing binding constants for Dnp, thenthe predicted response should be the sum of a number of individualcellular response curves. Each curve would be like that in FIG. 16, butthose with lower binding constants would be displaced to the right by anamount proportional to the ratios between their binding constants forDnp. Inspection of FIGS. 16 and 17 from this point of view indicatesthat the observed width of the experimental dose-response curves may beunderstood as resulting from the summation of responses from individualpopulations of cells having receptors differing in binding constants by1-1.5 log units—i.e., 10 to 30-fold. The dose-response measurements canbe fit within experimental error by summing the theoretical responses ofthree or four such populations.

For a constant dose of immunogenic polymer, Eq. 1 also can be used topredict the extent of reduction of response that will be obtained withdoses of increasing amounts of nonimmunogenic polymer N. Measurements ofthis type are shown in FIG. 18 for BALB/c mice. The solid line in FIG.18 is not fitted to the data but is calculated directly from Eq. 1 byusing the estimated value of the maximum-response dose D_(s) ^(max) of0.5 μg per mouse obtained from FIG. 17. The agreement between theexperimental points and the calculated theoretical curve in FIG. 18 isremarkable, if one considers the absence of arbitrarily adjustedparameters in this calculation.

In addition to experiments in living animals shown in FIGS. 16, 17, and18, dose-response curves were measured in vitro with isolated mousespleen cells. FIG. 19 shows the results of such an in vitro experimentas compared with a visually fitted theoretical curve calculated fromEq. 1. This agreement between experiment and theory for the in vitroexperiment with cultured spleen cells (FIG. 19) is approximately as goodas it was for the in vivo experiment with whole mice (FIG. 16). In bothcases, the measured response curve is somewhat broader than thatpredicted from a model based on a homogeneous hapten binding constant inthe responding cells.

Of particular significance to the present invention are measurements ofthe inhibition of immune response in vitro with increasing amounts ofnonimmunogenic polymer which are shown in FIG. 20. The solid line is notfitted to the data but is calculated directly from Eq. 1 by using thevalue of the maximum-response dose, D_(s) ^(max) of 0.4 ng/ml from FIG.19. There is substantial agreement between the experimental points andthe calculated theoretical curve.

The blood volume and extracellular fluid volume of a mouse are each ≈1ml, so the optimal immunogenic polymer does in vivo is ≈1 μg/ml. Thereis a large apparent discrepancy between this in vivo dose and that whichis optimally immunogenic in vitro (≈1 ng/ml). The almost 1000-foldsensitivity difference is largely explained by rapid removal in vivo ofpolymer molecules by phagocytes located throughout the body. Studieswith ¹²⁵I-labeled preparations of the polymers, as described in theabove-noted 1976 paper, showed that the bulk of the injected polymer isquickly removed from the circulation by Kupffer cells in the liver andphagocytic cells in other tissues. The resulting rapid fall in freepolymer concentration, coupled with uncertainties concerning the rate ofequilibration of polymer between different body fluid compartments makesdifficult any quantitative comparison of relative optimum concentrationsin vivo and in vitro. In spite of these difficulties, there remains thefact that the shapes of the dose-response and dose suppression curvesmeasured in vivo are remarkable similar to those measured in vitro,implying strongly that the same limiting process is being probed in bothcases. Furthermore, in both cases, the measured responses as a functionof dose are in excellent agreement with values obtainable from Eq. 1.

Although polymer N fails to stimulate at any dose, it inhibits polymer Sat the same dose where polymer S is maximally stimulatory. Thisindicates a competition for surface receptors. Because both polymerpreparations have almost identical “epitope densities” with a commoncarrier chemistry, this finding is in disagreement with theories thatexplain immunogenicity by invoking epitope density or polyclonal (i.e.,nonspecific) activation by the “carrier.”

Discussion of Example 6

The data presented above indicate the following with regard to aspecific T cell-independent stimulus: (i) a specific immunogenic signalis generated by the formation of immunons on the surface of a responsivecell, (ii) an immunon will form only after a sufficient number ofsurface receptors are clustered, and (iii) specific clustering ofsurface receptors occurs as a consequence of their being bound to linkedhaptens. This binding is specific for the hapten-receptor interactionand does not primarily depend on the “scaffolding” to which the haptensare attached. The underlying physical scaffold that links the haptensmay be molecular in nature or may consist of a surface on which smallhapten-containing structures are aggregated, as on the surface of an“antigen-presenting cell.”

Nonspecific stimuli, such as mitogens, lectins, antibodies against cellsurface proteins, and activating or inhibiting factors from other cells,may well influence the level of “irritability” of the responding cell,making it more or less likely to respond to a given amount ofimmunogenic signal or even to respond in the absence of specificsignals. Factors from T cells and macrophages have previously been shownto enhance antibody responses nonspecifically. Mitogens are known tostimulate cells nonspecifically to secrete antibodies. Whether or notthey do this directly or indirectly by a mechanism involving specificreceptor aggregation is not known. However, in contrast to thesenonspecific stimuli, the data herein indicates that specific stimulationoccurs by means of the linkage of receptors by their specific bindingsites into immunons; thus, cells displaying those receptors arestimulated to divide and differentiate into cells that will secretespecific antibodies.

It has been demonstrated above (and in the above-referenced 1976, 1982and 1983 papers) that molecules consisting of haptens linked to aflexible linear polymer are immunogenic only if they have a sufficientnumber of adequately spaced haptens. This finding with a Tcell-independent antigen might at first seem contradictory to the factthat many protein molecules that are T cell-dependent antigens and whichdo not contain multiple identical antigenic sites are never-thelessantigenic. However, several studies have shown that the antigenicity ofproteins in vivo depends on their state of aggregation. It is well-knownthat experimentally induced aggregation of protein molecules by physicalmethods (heat, adsorption to bentonite, emulsification with Freund'sadjuvant) or by chemical methods (cross-linking with glutaraldehyde, oralum) greatly enhances their antigenicity. Nonaggregated proteinmolecules centrifuged free of aggregates or collected from the sera ofinjected animals have been shown to be not immunogenic but tolerogenic,whereas aggregated material with presumed multiple antigenic sitesproduces an immune response. Therefore, it is possible that the minimumrequirements for antigenicity as determined with simple Tcell-independent polymer may have applicability to immune responses to alarge variety of molecules, including T cell-dependent ones. It is inany case evident that the suppressive effect of the nonimmunogenicpolymer, on the immunogenic polymer, as illustrated above, can be usedto control undesired immune response. The amount of nonimmunogenicpolymer so used will necessarily vary depending on the specific immuneresponse which is involved, the polymer carrier, the effective number ofepitopes involved, body weight and other factors. It is believed,however, that the administration of from 0.5 to 50 mg/kg body weightwould be effective in controlling undesired immune response. Theadministration may be effected by, for example, injection using asterile solution of the non-immunogenic polymer.

Example 7

Extension of Immunon Model to Alternative Haptens and Carriers

As is evident from the introduction and discussions above, the inventionis not dependent on the nature of the hapten or carrier but on themolecular mass of the carrier and the hapten density, these physicalcharacteristics (molecular mass, hapten density) determining whether ornot the matter is immunogenic or non-immunogenic or suppressive. This isfurther illustrated by the following additional disclosure andexemplification of tests done using fluoresceinated carriers. In thisfurther work, the molecular characteristics of five chemically differentfluoresceinated (Fl)-polymers were systematically varied, and theirability to stimulate an anti-hapten immune response was measured. Thepolymers used as carriers were carefully size-fractionated and consistedof one natural polymer (dextran), one modified natural polymer(carboxymethyl cellulose), and three synthetic polymers (Ficoll,polyvinyl alcohol, and polyacrylamide). The carriers varied in physicalstructure from the highly cross-linked Ficoll, to the somewhat brancheddextran to the linear polyacrylamide, carboxymethyl cellulose andpolyvinyl alcohol. Polymers were haptenated with Fl andsize-fractionated so as to yield a panel of molecules with varyingmolecular mass, hapten valence and hapten density. Anti-Fl response tothese haptenated polymers was measured in vivo after i.p. injection ofthe Fl-polymer in saline, and measured in vitro following culture withunfractionated spleen cells from naive mice.

In agreement with the foregoing exemplification involvingDnp-polyacrylamide, it was found that to be immunogenic, each of theFl-polymers had to exceed a comparable threshold value of molecular massand of hapten valence. Optimal immunogenicity occurred when theFl-polymers had values of mass and hapten density lying within apredictable range. Immunogenicity decreased when these optimalparameters were substantially increased or decreased. Accordingly, itcan be concluded that the immunogenicity of soluble haptenated polymersdepends on predictable physical molecular characteristics, and isrelatively independent of the chemical composition and conformation ofthe carrier polymer.

Polymer carriers selected to be haptenated were dextran (T 2000, T 500and T 70—Pharmacia); Ficoll (400 and 70—Pharmacia); carboxymethylcellulose (medium viscosity—Sigma); polyvinyl alcohol (average molecularweight 115,000—Aldrich); and linear polyacrylamide (synthesized inaqueous solution from crystalline acrylamide).

The polymer carriers were conjugated with fluorescein by the followingprocedures: Reactive carboxyl groups were generated in polyacrylamide bypartial hydrolysis in 0.05M Na₂CO_(3—)0.05M NaHCO_(3,) pH 10.1, at 20°C. (3). Amino groups were introduced into such deamidated polyacrylamideand also into dextran, Ficoll, polyvinyl alcohol and carboxymethylcellulose according to the procedures disclosed by Inman, J. Immunol.114:7044. Subsequently, the amino groups on the polymers were conjugatedto excess fluorescein isothiocyanate at pH 9.2 in 0.1M Na₂B₄O₇. Thepolymers were then dialyzed exhaustively against the buffer used forsubsequent gel filtration (0.1M NaCl, 0.001M EDTA, 0.02% NaN_(3,) 0.01MKPO₄, pH 7.4).

Fl-polymers were then repeatedly fractionated over 95 cm columns ofSepharose CL-2B, CL-4B and/or CL-6B; center cuts were taken repeatedlyto give preparations of relatively narrow molecular weightdistributions. Fl content was determined by measuring optical density at496 nm in 0.01 M Na₂B₄O₇ using a molar extinction coefficient of 72,000for Fl. This measurement together with polymer dry weight measurementpermitted calculation of epitope density. Molecular mass was determinedby sedimentation equilibrium analysis in the analytical ultracentrifugeas known in the art (Proc. Natl. Acad. Sci 73:3671 1976). Measurementswere performed at several polymer concentrations by using the shortcolumn method, and molecular mass was obtained by extrapolation to zeropolymer concentration. Polymers used in experiments were dialyzedagainst PBS and were sterilized by filtration with the use of 0.22-μmNucleopore filters.

For in vitro studies, suspensions of 2×10⁷ nucleated spleen cells fromnaive mice (CAF./J female mice, mostly 8-10 weeks old) were cultured ina final volume of 2 ml with or without appropriate polymer in 15 mlsterile polystyrene centrifuge tubes placed at an angle of 3 degreesfrom the horizontal. After 3 days of incubation, cells were harvestedand washed. Assay for direct (IgM) anti-hapten plaque-forming cells(PFC) was performed using a modification of the procedure described inTrans. Rev. 18:130 (1974). All cultures were done in triplicate and PFCassays were performed on each culture in duplicate. Immune response wasexpressed as PFC per 10⁶ spleen cells. Responses of control cultureswithout added antigen were subtracted from those of experimentalcultures. Typically, this control measured 5+/−2 PFC per 10⁶ cells.

Indicator cells in the plaque assay were hapten substituted at lowdensity in order to minimize assay response to low affinity (i.e.,non-specific) antibody. Substituted indicator cells were prepared bymixing 1 ml of packed burro red blood cells (BRBC) with a solution of 1mg of fluorescein isothiocyanate dissolved in 9 ml of borate bufferedsaline (BBS; 0.9% NaCl containing 10 mM sodium borate, pH 9.2). Themixture was then stirred for 1 hour at room temperature in the dark. Thecells were centrifugally washed first in BBS and then 3 or 4 times inPBS. They were stored in PBS containing 0.11% glycylglycine for nolonger than one week. They were washed in PBS before use. Fl-substitutedBRBC were found to be as effective as Fl-polymer substituted BRBC indetecting anti-Fl plaque forming spleen cells in this system.Trinitrophenyl (Tnp) substituted indicator cells were prepared asdescribed in J. Immunol. 131:2196 (1983).

In vitro studies were conducted in parallel with whole animalmeasurements in order to rule out possible differences in immunogenicbehavior due to differential body excretion rates or organ and tissuedistribution. Conversely, confirmation of in vitro findings by in vivoresults eliminated concern that in vitro findings merely reflectedartifacts of cell culture. Culture of unfractionated spleen cells wasthe in vitro assay of choice in order to mimic as closely as possiblethe cellular milieu to which these molecules might be exposed in theliving animal.

For in vivo antibody response, polymer preparations were injected intomice intraperitoneally in 0.5 ml isotonic saline. Adjuvants were notused in any antigen administration because they could change thephysical state of the antigen in such a way as to make interpretation ofactual molecular mass of the administered antigen impossible. After 4days, mice were sacrificed and their spleens removed for PFC assay.Responses of control mice injected only with saline were subtracted fromthose of experimental mice. Typically, this control measured 10+/−5 PFCper 10⁶ cells.

For the doses of Fl-polymers used to generate anti-hapten responses, nomore than 1% of the observed anti-Fl response could be generated whenunsubstituted carrier was used as immunogen. When tested fornon-specific polyclonal antibody generation, unhaptenated carriermolecules were found to generate no plaques against unsubstituted burrored blood cells (BRBC) or against BRBC substituted with eitherpneumoccoccal polysaccharide type 3 or with dinitrophenyl groups (datanot shown). These observations indicated that, in the doses used togenerate anti-Fl responses, Fl-polymers did not significantly stimulateB cells having epitope specificity distinct from fluorescein.

The composition and characteristics of the haptenated polymers usedherein are listed in Table 1 (see page 31).

All of these polymer carriers were essentially uncharged with theexception of the CMC which is negatively charged. Haptenation withfluorescein resulted in substituted polymers which were hydrophilic andnegatively charged.

It was found that the kinetics of response to this series of Fl-polymersclosely resembled those observed for Dnp-polyacrylamide. As an example,FIG. 21 shows dose-response curves of the primary in vitro anti-haptenresponse of naive spleen cells to Fl-PVA after various times ofincubation. The peak in vitro response occurred after three days ofincubation. The kinetics of the primary in vivo anti-hapten response tothe optimal dose of the same polymer are pictured in FIG. 22. Spleen PFCpeaked at about 4 days.

In vivo anti-hapten dose-response curves generated by four differentfluoresceinated polymers, Fl-Dex, Fl-Fic, Fl-CMC and Fl-PVA, are shownin FIG. 23. In vivo dose response curves, shown in FIG. 24, include thecurve generated by an additional polymer Fl-PA. These curves arerepresentative of the responses generated by all the immunogenicpolymers used in this study. Each dose-response curve is bell-shaped,initially increasing with the dose of antigen until a maximum isattained and then decreasing at higher doses of antigen.

Each of the size-fractionated polymers tested was consistent in behaviorin vitro and in vivo being either immunogenic or nonimmunogenic in bothsituations. Table 8 lists a number of representative polymers with theresults of assays for their stimulation of anti-hapten antibodyresponses.

TABLE 8 Immune Density Response Polymer (mM Fl/gm polymer) (in vitro^(a)in vivo^(b)) Fl₂₄₀Fic750 0.32 + + Fl₉₀Fic750 0.12 + + Fl₆₅Dex4000.16 + + Fl₆₀Dex170 0.35 +  N.D.^(c) Fl₉₅PA300 0.32 + N.D. Fl₂₃₀PA4000.58 + N.D. Fl₁₆₀CMC520 0.32 + + Fl₂₆CMC110 0.24 + + Fl₁₁₀PVA400 0.28 +N.D. Fl₅₅PVA200 0.28 + + Fl₁₄Fic40 0.35 − − Fl₆Fic35 0.17 − − Fl₁₄Dex400.35 − N.D. Fl₄₇PA80 0.59 − N.D. Fl₆CMC27 0.22 − − Fl₁₄PVA50 0.28 − −^(a))Determined by measuring direct anti-Fl PFC after 3 day culture ofnaive spleen cells with antigen. ^(b))Determined by measuring directanti-Fl-PFC of spleen cells harvested 4 days after i.p. injection ofantigen in saline without adjuvant. ^(c))N.D. = not determined

It is to be noted that the subscript number after the haptenabbreviation refers to the number of haptens per molecule (haptenvalence), while the number after the carrier abbreviations refers to themolecular mass in kD. For example, Fl₆₅Dex400 refers to a molecule with65 fluorescein groups on a dextran carrier, with a total molecular massof 400,000 daltons.

Over a 4 log dose range, the group of polymers listed above the dottedline were immunogenic and the group below the dotted line werenonimmunogenic. Both groups included molecules with each of the fivekinds of polymer carriers studied: Fl-Fic, Fl-Dex, Fl-PA, Fl-CMC andFl-PVA. Thus all five Fl-polymers have the potential to be eitherimmunogenic or nonimmunogenic, irrespective of the chemical compositionof the polymeric carrier. Examination of the molecular characteristicsof the polymers in Table 8 indicates that immunogenicity is directlyrelated to the molecular mass and the hapten valence. All polymers abovethe dotted line, had a hapten valence greater than 20 and a molecularmass larger than 100,000 daltons and were immunogenic. Polymers belowthe dotted line had a molecular mass less than 100,000 daltons and werenot immunogenic at any dose tested. The hapten densities in both groupshad approximately the same range: between 0.12 and 0.59 millimoles offluorescein per gram of polymer. Thus, hapten density by itself was nota predictor of the presence or absence of immunogenicity.

Example 8

Antigen Specific Suppression Independent of Carrier Chemistry

The inhibiting properties of nonimmunogenic Fl-polymers are furtherillustrated by the following example.

As shown above, soluble fluoresceinated polymers with molecular massunder 100,000 daltons and with hapten valence under 20 were unable tostimulate an anti-hapten response at any measured dose. However, thisexample shows that when mixed with optimal concentrations of stimulatoryFl-polymers and cultured with naive spleen cells in vitro, anti-haptenantibody production can be inhibited. FIG. 25 shows a representativeexample of such inhibition.

Naive spleen cells were cultured with a series of solutions formulatedto contain increasing concentrations of the nonimmunogenic polymerstogether with a constant concentration of the immunogenic polymerFl₉₀Fic750. As can be seen, the inhibitory ability of the nonimmunogenicpolymers increases with increasing concentration until completeinhibition of the anti-Fl response to the immunogenic polymer is reachedat inhibitor concentrations between approximately 1 and 10 ng per ml.

FIG. 25 demonstrates “cross-inhibition”, whereby Fl on the backbonecarriers, PVA, Dex, or CMC can inhibit the anti-Fl response stimulatedby Fl-Fic. The data indicate that the inhibitory potentials of thesenonimmunogenic Fl-polymers are largely independent of specific carrierchemistry. As a control, FIG. 25 shows that the irrelevant hapten, Dnp,on a PA carrier could not inhibit the anti-Fl response.Carrier-independent inhibition is further evidenced in Table 9, wherethe ability of four nonimmunogenic Fl-polymers to inhibit the immuneresponse to four immunogenic polymers with different carrier backbonesis shown.

TABLE 9 Carrier Independent Inhibitory Ability of Fl-Polymers InhibitoryHapten Density Concentration^(a) (ng/ml) for 50% Inhibition of Responseto Polymer (mM Fl/gm polymer) Fl₉₀Fic750 Fl₆₅Dex400 Fl₁₁₀PVA400Fl₁₀₅CMC440 Fl₁₄Fic40 0.35 0.5 1 1 0.3 Fl₁₄Dex40 0.35 0.35 2 N.D.^(b)N.D.^(b) Fl₁₄PVA50 0.28 0.4 3 0.5 N.D.^(b) Fl₆CMC27 0.22 0.4 2 1 1^(a)concentration giving 50% inhibition was determined by measuring thedecrease of direct anti-Fl PFC caused by adding the inhibitory polymerto a culture containing a constant amount of immunogenic polymer.^(b)N.D. = not determined

FIG. 26 shows inhibition curves generated by five chemically differentFl-polymers when mixed in increasing amounts with a constant amount ofFl₉₀Fic750. One of the curves illustrates the self-inhibition caused byadding increasing amounts of Fl₉₀Fic750 to an optimally immunogenicconcentration of the same polymer. For each of the Fl-polymers used,inhibition increases with dose. Although this may be termed “high-dose”inhibition, the actual in vitro molar concentration of inhibitornecessary for 50% inhibition of the response to Fl₉₀Fic750 did notexceed 30 pM for any of the Fl-polymers, and for Fl₁₀₅CMC440, it was aslow as 2 pM.

The influence of hapten density and molecular mass individually oninhibitory ability was also measured. Table 10 compares the inhibitoryabilities of pairs of Fl-polymers with similar molecular mass, butdiffering hapten densities. In each pair of molecules where themolecular mass was kept constant, the polymer with the higher haptendensity was the better inhibitor, i.e., lower concentrations wererequired to cause a 50% inhibition of the response to Fl₉₀Fic750.

TABLE 10 Effect of Hapten Density on Inhibitory Ability Conc.^(a) for50% Inhib. of Hapten Density FL₉₀Fic750 (mM FL/gm Response InhibitoryPolymer polymer) (ng/ml) (pM) FL₂₄₀Fic750 0.32 5 7 FL₉₀Fic750 0.12 25 33FL₂₃₀PA400 0.58 2 5 FL₆₅Dex400 0.16 10 25 ^(a))Concentration giving 50%inhibition was determined by measuring the decrease of directanti-Fl-PFC by adding the inhibitory polymer to a culture containing aconstant amount (3 ng per ml) of Fl₉₀Fic750.

Table 11 compares the inhibitory abilities of two sets of polymers, oneset with CMC as the carrier, and the other set with Ficoll as thecarrier. The hapten densities in each set are similar, but the molecularweights differ. Included in the CMC carrier set are two nonimmunogenicpolymers (Fl₆CMC27 and Fl₄CMC15); one nonimmunogenic polymer (Fl₁₄Fic40)is included in the Fic carrier set. In each set, regardless ofimmunogenic potential, the polymer with the higher molecular weight isthe better inhibitor.

TABLE 11 Effect of Molecular Mass on Inhibitory Ability Hapten DensityConc. (pM)^(a) for 50% Inhib. Inhibitory Polymer (mM FL/gm polymer) ofFL₉₀Fic750 Response FL₁₀₅CMC440 0.24 2 FL₂₆CMC110 0.24 9 FL₆CMC27 0.2215 FL₄CMC15 0.27 40 FL₆₄₀Fic2000 0.32 4 FL₂₄₀Fic750 0.32 6 FL₁₄Fic400.35 9 ^(a))Concentration giving 50% inhibition was determined bymeasuring the decrease of direct anti-Fl-PFC by adding the inhibitorypolymer to a culture containing a constant amount (4pM) of Fl₉₀Fic750.

Example 9

A. Suppression of Ongoing T-cell Dependent Immune Response Against aHapten

Very strong T-cell dependent responses against haptens can be raisedagainst haptenated proteins, such as hen egg ovalbumin (OVA) or bovineserum albumin (BSA), when these haptenated proteins are absorbed onaluminum hydroxide and repeated small injections are given. The responsethat results may contain high levels of both IgG and IgE antibodiesdirected against the hapten which is coupled to the injected protein. Asan example, the serum anti-fluorescein IgG response levels of threeindividual mice, which had been immunized by this protocol withfluorescein substituted OVA over a time period of several months andthen were followed for a number of weeks without further exposure toantigenic material, is shown in FIG. 27. These mice were part of a largecohort which had all been immunized simultaneously according to the sameprotocol. Some of these mice were then injected intraperitoneally withpolymers which we had previously determined were inhibitory. Suchpolymers were soluble fluoresceinated polymers of high haptensubstitution density, but with molecular weights under 100,000. Thesepolymers were injected to test their ability to suppress an ongoing highlevel anti-fluorescein IgG antibody response (cure). The results fromthe injection of three different such polymers on the serum levels ofindividual mice are shown in FIGS. 28, 29 and 30, where the time scaleof bleedings is the same as in FIG. 27.

The data in FIG. 28 show that, unlike the unsuppressed mice shown inFIG. 27, the mice which received a 3 mg does of a multiple FLsubstituted FL-Pa, FL30Pa50, had strongly diminished serum anti-Fl IgGantibody level for a period of a month or more. Of the 6 suppressedmice, the serum antibody levels of 5 mice fell quickly to levels too lowto measure and remained so for the entire period. The serum antibodylevel in the sixth mouse fell more slowly and showed a slight recoveryat the end of the time period.

When the polymer was dextran, FIG. 29, a-dose of 0.1 mg of FL25Dex70 hadno apparent effect on the serum anti-FL IgG antibody level, whereas asubsequent dose of 1 mg caused total suppression in 5 of the mice. Thesixth mouse showed a sharp drop to a low level, followed by a slow andsteady decline thereafter. The data in FIG. 30 show that a dose of 1 mgof FL30Dex80 caused a very substantial, but not total, suppression ofthe serum level of anti-FL IgG antibody. However, a subsequent dose of 3mg brought about total suppression.

From the combined data of FIGS. 27, 28, 29 and 30, it is apparent thatthe injection of milligram quantities of appropriate haptenated polymersinto an immunized mouse can cause profound and prolonged suppression ofthe level of serum anti-hapten IgG antibodies. This can be consideredequivalent to the “cure” of an established humoral immune response.

In a subsequent experiment, an effort was made to test for thesuppression (cure) of a high level anti-fluorescein response by theparallel measurement of the serum levels of both IgG and IgE (reaginic)antibodies, as well as the determination of the number of spleniclymphocytes producing anti-fluorescein antibody of the IgG class. Inorder to measure these different indices of suppression in the sameexperiment, a large number of mice were stimulated simultaneously withFL-OVA on aluminum hydroxide and subsequently subjected to differentprotocols of suppression and restimulation.

Very strong immune responses against the hapten, fluorescein, wereraised in a large group of CAF1 mice by repeated injection of variousdoses of OVA which had been chemically substituted with fluoresceinisothiocyanate to the level of 4.5 fluorescein hapten groups perovalbumin molecule. In order to generate a strong and uniform immuneresponse, the FL-OVA was adsorbed onto the adjuvant, aluminum hydroxide,Al(OH)₃, in the ratios of 0.1, 1, or 10 μg of FL-OVA per mg of aluminumhydroxide. A quantity of the resulting antigenic preparation containing1 mg of Al(OH), was injected intra-peritoneally into a series of mice inorder to bring about a strong antibody response against the fluorescein.After the second injection of antigen, the resulting T-cell dependentanti-FL IgG antibody level in the serum was uniformly very high.

In order to measure the resulting anti-FL IgG antibody level, it wasfound necessary to dilute the serum 100,000 fold so that quantitativemeasurements could be made by ELISA technique. Measurement was made on96 well plates coated with fluoresceinated gelatin, using affinitypurified, alkaline phosphatase coupled anti-mouse IgG (μ chain) secondantibody and optically following the rate of hydrolysis of nitrophenylphosphate. As shown in FIGS. 31-33, anti-FL IgG measurements were madeusing a 100,000 fold serum dilution, with three mice per point, showingaverage and standard deviation of the measurements.

An effort was made to determine the generality of the observations byvarying the stimulatory dose of FL-OVA over a 100 fold range, i.e., 0.1,1, or 10 μg of FL-OVA on 1 mg of aluminum hydroxide were injected. A lowlevel of fluorescein substitution on the gelatin which coated the ELISAanalysis plates was used in order to emphasize the better binding,higher affinity, more clinically relevant anti-FL antibody molecules.

The elimination or “cure” of the resulting strong immune response wasaccomplished by the injection of an adequate dose (2 mg) of anon-stimulatory, but inhibitory, fluoresceinated polymer. The particularpolymer used in this experiment was fluoresceinated dextran containing30 fluorescein groups substituted on dextran with an average molecularmass of 80 kDa for resulting polymer (FL30-Dex80) as determined by highpressure liquid chromatography analysis. Since the mice had highcirculating levels of antibody against fluorescein at the time they were“cured”, it was important to administer the curative dose in steps overan extended time interval in order to avoid undesirable side reactionsin the recipient animals. Accordingly, doses were increased a maximum of10 fold every 2 hours beginning with an initial dose of 1 μg. Thisprotocol produced no visual evidence of distress in the animal during orfollowing the administration of the FL-Dextran. Cure doses wereadministered on day 35 when the immune response was very substantial,and also on day 95, following the attempt to restimulate the animals onday 75.

A direct comparison of the responses of the mice which had beenstimulated repeatedly, with mice which had been stimulated and thencured, is shown in FIGS. 31, 32 and 33. These Figures show the dramaticdecrease in the anti-FL antibody levels following the cure dose, as wellas the lack of response to a subsequent restimulation on day 75. Inorder to demonstrate the range of effectiveness of the cure treatment,these Figures include data from mice which had been previouslystimulated with high (10 μg), medium (1 μg) and low (0.1 μg) doses ofthe stimulatory antigen, FL-OVA on aluminum hydroxide. FIGS. 31, 32 and33 clearly show that the serum anti-FLU IgG antibody level is verysubstantially reduced by a single cure treatment in each case, and isnot restimulated by repeating the original process of stimulation.

In addition to the measurement of the level of circulatinganti-fluorescein IgG antibody molecules in the blood, it is important toknow the number of lymphocytes which are actively secreting suchmolecules into the serum. Ideally, suppression of an ongoing antibodyresponse should cause a decrease in both the level of circulatingspecific antibody and also in the number of lymphocytes which secretesuch molecules. The spot ELISA plaque assay method (Greene, G., et al.,J. Imm. Methods, 129: 187-197, 1990) allows the measurement of thenumber of-spleen cells which are actively secreting IgG antibodyspecific for fluorescein.

When this method was used at 125 days to analyze the pooled spleen cellsfrom 3 mice which had been stimulated three times (at 0,21 and 75 days)with different amounts of FL-OVA on aluminum hydroxide, substantialnumbers of cells were found to be secreting anti-FL IgG antibodies ineach case, FIG. 34, (“non-cured” bars). However, when cells wereanalyzed from the spleens of mice which had been stimulated by the sameprotocol, but which had also been cured on days 35 and 95 as discussedabove, very substantial reductions in the number of cells producinganti-FLU IgG antibody molecules were found in each case, FIG. 34,(“cured” bars). These data indicate that the cure process not onlyeliminated the relevant free circulating antibody from the serum, butalso, eliminated most of the spleen cells capable of producing suchantibody molecules. From the relative magnitudes of these twomeasurements the degree of inhibition of antibody producing cell causedby the suppression with FL-Dex was calculated and the resulting valuesare shown in FIG. 35.

The data shown above indicate that very substantial suppression of thespecific immune response occurred as a result of the treatment withsuppressive FL-Dex. From the data in FIG. 35, as well as additional datanot shown, two different trends can be discerned relating the measureddegree of suppression with the experimental conditions:

1. The measured suppression increases as the dose of immunogen whichproduced the immune response decreases, and

2. The measured suppression increases as the amount of epitope(fluorescein) coupled to the gelatin used for the spot-ELISA assaydecreases, (i.e. the percent inhibition measured with 0.08 FL pergelatin is larger than when measured with 0.2 FL per gelatin). Moremeasurements (data not shown) have indicated that this trend holds trueacross a wide range of hapten substitution density in the assays forcells producing anti-Fl antibodies in both the spot-ELISA assay forantibody-producing cells and the ELISA assay for serum levels of IgGantibody.

The two trends discussed above are consistent with the conclusion thatthe antibodies whose occurrence is most effectively inhibited by thesuppressive polymers discussed above are those with the highest affinityfor the hapten (FL). Since the correlation of allergy and autoimmunedisease states with the presence of small amounts of high affinityantibody has often been made, this is a promising observation forapplication to medically relevant situations.

B. Cure of High Level Anti-FL IgE Response

Many of the effects of allergy are due to the presence of antibodies ofthe IgE class, which can activate histamine releasing mast cells whenexposed to the relevant antigenic material. A highly specific biologicalassay for the presence of such antibodies of the IgE class is thepassive cutaneous anaphylaxis (PCA) test, wherein a few microliters ofdiluted serum from the animal under analysis is injected into the skinof a test animal. An hour or two later the test animal is injectedintravenously with the relevant antigen in a saline solution containingsoluble dye. In the skin regions where injected serum IgE antibodiesagainst the antigen are present, visible dye color appears as a resultof the activation of mast cells with subsequent release of mediatorcausing vascular leak. The greatest dilution of injected serum whichwill provoke an observable skin response is a measure of the IgEantibody titer in that serum. This is usually detectable down to a levelof nanograms of the specific IgE antibody per ml of undiluted serum.When this biologically significant PCA test was applied to the serumfrom some of the mice which were stimulated and suppressed as shown inFIG. 32 above, the results shown in FIG. 36 were obtained.

FIG. 36 demonstrates that, after substantial levels of IgEanti-fluorescein antibody had been developed, a single injection ofsuppressive Fl-Dex brought the serum level of such IgE antibody to avery low level for several weeks (actually, the level was so low that itwas experimentally indistinguishable from the background level of themeasurement). Furthermore, the mice so treated were completely resistantto boosting with a repeated dose of antigen, whereas the control miceshowed a very substantial increase in their IgE serum titer whenboosted. It appears that the suppressive polymer injection caused a longlasting “cure” of an established allergic type response in the mice.

In summary, experimental tests of the ability of suppressive forms offluoresceinated polymers to suppress or “cure” a strong ongoing T-celldependent immune response against the fluorescein hapten have been made.The results indicate that clinically relevant IgG and IgE antibodiesspecific for the hapten can be effectively eliminated, as measured bydramatic and long lasting reductions in:

1. Serum anti-hapten IgG antibody level;

2. The number of splenic lymphocytes secreting anti-hapten IgG antibody;and

3. Serum anti-hapten IgE antibody level.

C. Reduction of Serum Anti-Penicillin IgE

The penicillin allergy is among the most clinically distressing drugallergy since administration of penicillin (or its related compounds) isstill the treatment of choice for many diseases. However, many peopleare allergic (i.e. show immediate-type hypersensitivity reactions) topenicillin or become so while undergoing long-term penicillin therapy.Described below are the data that indicate that the immune response topenicillin can, in fact, be specifically suppressed using thistechnology.

The experimental details that follow apply to the study set forth inthis Example.

Animals: CAF, (BALB/c X A) female mice were obtained from CumberlandFarms, Clinton, Tenn. and were approximately 10 weeks old when firstimmunized. Male Sprague-Dawley rats weighing 320-380 g were obtainedfrom Holtzman Co., Madison, Wis.

Chemicals: Bovine serum albumin (BSA) Fraction V was obtained from MilesLaboratories, Inc. Penicillin G (sodium salt) and crystallized chickenovalbumin (OVA) were obtained from Sigma Chemical Corporation, St.Louis, Mo., p-Chloromercuribenzoate (PCMB) was obtained from Calbiochem,Los Angeles, Calif., Evans Blue was purchased from Eastman Kodak,Rochester, N.Y. and ethylene diamine (EDA) was ordered from MCBManufacturing Chemicals, Cincinnati, Ohio.

Hapten-Carrier Conjugates: Benzylpenicilloyl-bovine serum albumin(BPO-BSA) and benzylpenicilloyl-ovalbumin (BPO-OVA) were prepared byincubating BSA or OVA with Penicillin G (benzylpenicillin) in 0.5 MK₂CO_(3,) pH 10.0 at room temperature (Nakawaga et al, Int. Archs.Allergy Appl. Immunol. 63:212 (1980)). Various incubation times yieldeddifferent epitope densities. The number of haptens per carrier isdenoted by subscript, i.e. OVA substituted with four BPO groups isBPO₄-OVA. The degree of substitution was determined by a modification ofthe penamaldate assay (Parker, C. W. Methods in Immunology andImmunochemistry, Williams and Chase eds. Vol. I, p. 133, Academic Press,N.Y. (1967)). 0.1 ml of 2×10⁻³ M PCMB in 0.05 M carbonate, pH 9.2 isadded to 1.0 ml of the penicilloyl-carrier conjugate in 0.05 Mcarbonate, pH 9.2. The approximate penicilloyl concentration should be 2to 4×10⁻⁵ M. After mixing and allowing to stand at room temperature for5-10 minutes, a reading is made at 285 mμ (ε=2.38×10⁴). The incrementalincrease, after correction for uncombined PCMB (0.038 at a finalconcentration of 2.82×10⁻⁴ M) and dilution (protein concentration is 91%of original) is due to the penamaldate formed from PCMB and thepenicilloyl group. Benzylpenicilloyl-polyacrylamide (BPO-PA) was made bycarefully size-fractionating linear PA by gel filtration as describedabove. Incubating the PA with ethylene diamene (EDA) at 50° C. for 90minutes followed by extensive dialysis results in the formation ofheavily substituted EDA-PA. This was then conjugated with penicillin bythe method used to prepare the protein conjugates. The resultinghaptenated polymer was then refractionated by gel filtration to relativesize homogeneity. (See FIG. 37)

Immunizations: Two groups of four mice each were injectedintraperitoneally (i.p.) with 1 μg of BPO₄-OVA on 1 mg Al(OH)₃ in 0.10ml of 0.01 M Tris, 0.15 M NaCl buffer, pH 8.3. A booster injection of 3μg BPO₄-OVA on 1 mg Al(OH)₃ was given two and one-half weeks after theprimary injection. Approximately three and one-half months after theprimary immunization, the mice were re-challenged with 3 μg BPO₄-OVA on1 mg Al (OH)₃.

Suppression: One of the above groups of four mice received an i.p.injection of 1 mg of BPO-PA in 0.25 ml phosphate buffered saline (0.01 Mphosphate, 0.15 M NacL, pH 7.4 PBS). This polyacrylamide had anapproximate molecular weight of 40,000 (determined by equilibriumultracentrifugation) and was substituted with approximately 25 BPOgroups per polyacrylamide molecule. Prior studies have shown thispreparation of BPO-PA to be non-immunogenic at any dose.

Assay: IgE content was determined by a modification of the passivecutaneous anaphylaxis (PCA) assay (Ovary, Int. Archs. Allergy Appl.Immun. 3:293 (1953)). Equal volumes of serum from mice in each groupwere pooled and 0.1 ml volumes of diluted serum were injected into theskin of rats. After two hours, 4 mg BPO₈-BSA plus 10 mg Evans Blue Dyein 0.5 ml PBS was injected intravenously (i.v.) and twenty minutes laterthe rats were sacrificed, skinned, and the titer (the reciprocal of thehighest dilution yielding a lesion at least 5 mm in diameter) wasdetermined.

Mice injected with BPO₄-OVA on Al(OH)₃ gel developed an anti-BPO IgEresponse (FIG. 38) as measured by PCA assay. This anti-BPO response isthe murine correlate of human penicillin allergy. Four mice wereinjected with a suppressive dose of 1 mg of the polyacrylamidehaptenated with BPO (BPO-PA). Within one week of receiving thesuppressive dose of BPO-PA, serum levels of anti-BPO IgE in theexperimental group declined by greater than 98% (FIG. 38a) while thelevels of anti-OVA IgE remained constant (FIG. 38b). That is, theresponse of the experimental group to the BPO hapten, after suppression,was less than {fraction (1/80)} of the control group response.

Approximately two and one-half months after the experimental groupreceived its suppressive dose of BPO-PA, both groups were boosted withan i.p. injection of 3 μg of BPO₄-OVA on 1 mg Al(OH)₃. The mice in thecontrol group had an anti-BPO response even greater than the originalresponse while the mice in the experimental group were unresponsive tothe “boosting” injection (FIG. 38a). Therefore, the suppression inducedby the BPO-PA is not only fast (within one week), but lasts severalmonths. Furthermore, it tolerizes the mice so that they are unresponsiveto additional exposure to the BPO hapten.

D. Use of Valence-restricted Cyclodextrin Based Conjugates asSuppressive Constructs

Balb/c mice were immunized and subsequently boosted twice with Fl-BSAadsorbed on to aluminum hydroxide to raise high titre IgG anti-Flantibodies. These mice were divided into groups and treated either withthe valence-restricted scaffold bearing seven FITC groups (CI-0374) asdescribed in Example 1H above at three different doses or with dextranof 70,000 dalton substituted with 60 FITC groups (CI-0323) at a doseshown in previous experiments to be optimally suppressive. Another groupwas immunized with the buffer alone as a control. Mice were bled atintervals following these treatments and sera were assayed by ELISA forIgG anti-Fl antibodies as described in previous Examples. It is apparent(see FIG. 39) that the valence-restricted scaffold can inducedose-dependent, long-lasting suppression of the anti-FL response similarto that induced by the Fl-dextran construct.

Example 10

T-Cell Dependent Antibody Responses to Proteins and Protein Oligomers

An assumption of the Immunon model of immune responsiveness is thatmonomeric protein molecules, which contain only a single copy of eachkind of potential epitope, should not be immunogenic if administered inmonomeric soluble. form. However they may be immunogenic if administeredin polymerized form or if they are polymerized into closely spacedarrays absorbed on adjuvants, on cell surfaces, as soluble or insolubleaggregates, or by some other process within the body.

Immunogenicity of Polymeric BSA: Measurement of Anti-BSA IgM

The results obtained from the study of polymerized BSA will be describedfirst.

As was the case with the previous haptenated dextran studies, theanti-BSA IgM serum levels were found to rise rapidly, peaking at about 6days and then declining to a plateau level. FIG. 40 shows that solublehighly polymerized BSA (a “70-mer,” containing 70 BSA monomers) iscapable of raising IgM antibodies even at very low doses, whereasmonomeric BSA requires substantially higher doses to bring up detectableIgM levels against BSA.

When data from a series of polymers of BSA of differing molecular weightis compared, in FIG. 41, the immunogenicity, as measured by IgM levelsat day 6, is found to increase most rapidly at the higher molecularweights, but is strongly dose dependent at all molecular weights.

Immunogenicity of Polymeric BSA: Measurement of Anti-BSA IgG

By day 14 after the injection of the soluble polymers of BSA,substantial isotypic class switching had occurred and anti-BSA IgGantibodies were found to be present for some combinations of antigendose and molecular mass, FIG. 42. When multiple small injections of BSApolymers were given, the response was very dependent upon the molecularweight of the BSA polymer. FIG. 43 illustrates experiments in which micewere given three injections 30 days apart of 1, 10, or 100 μg of BSApolymers in saline. As was true for single injections, the anti-BSA IgGserum levels were strongly dose and polymer size dependent. FIG. 43indicates that monomeric BSA is not very effective in producing ananti-BSA IgG response even after repeated injections in saline at dosesup to 100 μg. In contrast, preparations containing polymers ofsubstantial size were very effective. This observation was confirmedwhen the total number of consecutive injections, on a monthly basis, wasincreased to five, as is illustrated in FIG. 44. FIG. 44 illustratesmore clearly a trend which was evident in the previous Figures, i.e.,that significant amounts of antibody are raised only to the polymericform of soluble BSA when small doses are administered. It is also clearthat even very small doses (1 μg) of highly polymeric protein can beimmunogenic in the absence of adjuvant, if they are administeredrepeatedly.

Immunogenicity of Polymeric OVA: Anti-OVA IgM

Results which were very similar to those obtained with polymerized BSAwere obtained when polymerized ovalbumin, OVA, was used as antigenicmaterial. The primary response to monomeric and highly polymerized OVA,both injected in saline, is shown in FIG. 45.

Immunogenicity of Polymeric OVA: Anti-OVA IgG

When glutaraldehyde polymerized OVA was size fractionated and severaldifferent doses of the individual fractions were injected three times atmonthly intervals, the immune response was found to depend strongly onthe OVA-polymer size and the dose, FIG. 46 The variations of responsewith dose and polymer size are roughly comparable to those found withpolymers of BSA, FIG. 42.

A comparison of the primary IgM responses to 1 mg of either polymerizedBSA or polymerized OVA, at short times after administration of antigen,FIG. 47, shows a substantial degree of similarity. Both Figures showincreasing immunogenicity with increasing polymerization.

Anti-fluorescein Response Generated By Fluoresceinated BSA Polymers

In order to determine the immune response to a hapten on polymerizedprotein, fluorescein was coupled to BSA polymer at a number of differentlevels of substitution, and the immune response was determined afterseveral injections, as shown in FIG. 48.

The results demonstrate that the anti-hapten response was of the desiredIgG isotype. It rose with increasing degree of substitution, peaking atapproximately 5 fluoresceins per BSA monomer unit, or a total of 100fluoresceins per BSA 20-mer. It then fell rapidly to very low levelswith increasing substitution. On the other hand, the immune response tothe BSA itself remained relatively constant with increasing fluoresceinsubstitution until approximately 5 haptens had been added, whereupon it,rather surprisingly, also fell rapidly. This indicates that there isalso an optimum level of substitution of polymerized proteins withpeptide epitopes of types potentially useful for vaccines. Constructionof maximally immunogenic adjuvant-free vaccines using this type ofchemistry is contemplated.

It can concluded from the foregoing results that:

1) Polymeric BSA and OVA, administered without adjuvant, stimulateconsiderable IgM and IgG anti-protein responses.

2) The immunogenicity of these poly-proteins increases with increasingprotein multiplicity.

3) Immunogenicity of poly-proteins is strongly dose-dependent, theimmunogenicity increasing with increasing dose.

Example 11

Suppression of Antibody Responses to Peptides from Extrinsic Antigensand Autoimmune Antibody Responses Against Epitopes on EndogenousProteins

The following materials and methodologies are referenced in thedescription of experimental results that follows:

Mice—Balb/c femal mice were obtained from either the Jackson Laboratory,Bar Harbor, Me. or Harlan/Sprague Dawley, Indianapolis, Ind. They wereused at 8-10 weeks of age.

Immunization protocols—To raise IgG antibodies (Abs), mice were given asingle intraperitoneal injection of 10-50 μg peptide-BSA conjugateadsorbed on aluminum hydroxide. Test bleeds were taken at various timesthereafter and anti-peptide IgG Ab titers measured. To test theimmunogenicity of peptide-dextran conjugates, mice were injectedintraperitoneally with 100 μg doses of dextran backbone to whichpeptides were conjugated at various substitutions ratios. Bleeds weretaken at weekly intervals and levels of IgM and IgG peptide-specific Absin the sera were measured (FIG. 49). The suppressive peptide-dextranconjugates were administered in the following way unless otherwiseindicated: 1, 10 and 100 μg doses were injected at 2-hourly intervals,with the 1 and 10 μg doses being given intravenously whereas the highdose was given intraperitoneally (“cure” treatment). Subsequent doses tomaintain suppression were given at weekly intervals.

ELISA assay—Antibody titers were measured by standard solid-phase ELISAassay. Microtiter plates (Immunolon II, Dynatech Labs, Alexandria, Va.)were coated overnight at 4° C. with peptide-gelatin conjugates at 0.1μg/well. After blocking plates with PBS/gelatin, various dilutions ofantisera were added and incubated at room temperature for two hours.Plates were washed and antibody binding was detected with horseradishperoxidase-conjugated isotype-specific antibodies (Kirkegaard and PerryLabs, Gaithersburg, Md.) followed by the ABTS substrate. Data areexpressed as OD_(403nm) of the ABTS product. Antibodies directed againstlinker regions were detected using an irrelevant peptide-gelatinpreparation and these readings were subtracted from those for specificbinding.

The first peptide chosen for study was a sixfold repeat of a glutamicacid-alanine-leucine-alanine SEQ ID No:20 sequence (EALA using singleletter amino acid code) followed by the peptide sequenceglycine-alanine-glycine-arginine-glycine-asparticacid-serine-proline-alanine-amide SEQ ID No:21. This peptide will bereferred to herein as (EALA).

Defined EALA-dextran conjugates were synthesized on 84,000 MW Dextran(EALA-Dex₈₄) that were verified to be non-immunogenic. Using theseconjugates, the ongoing anti-EALA IgG response elicited by EALA-BSA wassuppressed. Forty-six days after injection of EALA-BSA, these mice weresplit into two groups one of which received EALA-dex₈₄ in increasingdoses of 1, 10 and 100 μg dextran backbone and the other of whichreceived three injections of buffer alone. The mice were bled three dayslater and at three day intervals thereafter and the antisera tested forany reduction in their anti-EALA IgG titers. FIG. 50 clearly indicatesthat EALA-dex₈₄ prevented the rise in Ab levels that continued toprogress in the untreated mice. The low levels persisted for at least 21days. At this point another cure was performed using the same regimen tosee if the low response of suppressed mice could be effectivelyabolished altogether. Although the response was not reduced further byEALA-dex₈₄ administration, it was maintained at low levels for at leastanother 14 days while that of the control mice still appeared toincrease.

For the second study a different peptide was chosen that has beenstudied extensively by the immunologic community. This peptide, referredto as 159, represents residues 331-339 of chicken ovalbumin (OVA). Thepeptide consisting of residues 323-339 of this protein, referred to hereas 104, is a dominant epitope on OVA recognizable by helper T cellsparticularly in H2^(d) mice.

When 104 is arrayed on dextrans of high molecular weights, and theseconjugates are injected into Balb/c (H2^(d)) mice, high IgG Ab responsesare rapidly obtained. In addition, when 104 was arrayed on BSA,precipitated with aluminum hydroxide and injected into mice, anextremely vigorous antibody response was seen. Interestingly, it wasfound that approximately 40-50% of the antibody response to 104, eitherarrayed on dextran or BSA could be attributed to the C-terminal 10 aminoacids represented by 159. This system then is analogous to a morecomplex antigen (such as a protein) wherein a short, linear sequence ofamino acids is recognized by a significant proportion of the antibodiesgenerated to the entire antigen.

Using this model, the ability to specifically and selectively suppress aresponse to a defined epitope of a more complex system was demonstrated.Initial studies designed to better characterize this system revealedthat peptide 159, when arrayed on 65,000 MW dextran was not immunogenic,as predicted by the Immunon model. However this peptide can berecognized by B cells since BSA conjugates raise good antibody responsesto 159. Furthermore, as mentioned above, a substantial portion ofantibodies raised by the 104-conjugates are directed against 159 asindicated both by direct binding in ELISA assay and by competition assayusing free 159 to inhibit binding of anti-104 antibodies to 104-gelatin.

FIG. 51 demonstrates the ability to suppress specifically the portion ofthe 104 response that was directed towards the 159 epitope(s). IgGantibodies were raised to 104 by injection of Balb/c mice with 104-BSAadsorbed on to alum. High titers were raised both to 104 and to 159(FIG. 51). Forty days after the immunizing injection (day 0 in theFigure), groups of eight mice were either injected with saline or with1, 10 and 100 μg dextran of 40,000 molecular weight conjugated with 159at a ratio of 10 moles peptide/mole dextran (ie 159₁₀-dex₄₀). Mice weresubsequently bled at day 3 and then at 7 day intervals and assayed forresponses to both 159 and 104. FIG. 51 shows the responses after thecure and indicates that the response to 159 was dramatically reducedimmediately after treatment but that the antibody titer reboundedvirtually to control levels within 14 days. The response to 104 intreated mice followed the same pattern of reduction and recovery as the159 response but was only reduced at day 3 to about 60% of the precureresponse whereas the 159 response is reduced to about 9% of precurelevels. In other words, approximately 40% of the response to 104 isremoved by administration of 159₁₀-dex₄₀ indicating that this portion ofthe 104 response is directed against epitopes in the 159 sequence. Infact, up to 40% of the 104 response can indeed be completed successfullywith soluble 159 in solution phase competition assays (data not shown).

The cure was then repeated as before but using 200 μg as the largestdose. In addition, 200 μg doses were given at weekly intervalsthereafter to see if continuous administration of suppressive conjugatewould maintain a chronic suppression of the 159 response. FIG. 51 showsthat this second cure again essentially completely inhibited theresponse to 159 measured 7 days later and this was comparable to thereduction seen after the first cure (the response was 13% of precurelevels 7 days after the first cure and 10% of precure levels 7 daysfollowing the second cure).

Furthermore, the response to 159 remained suppressed over the next 40days during which 159₁₀-dex₄₀ was injected intraperitoneally at weeklyintervals. Injections were then stopped and antibody responses weremonitored for approximately 30 more days and were found to staysuppressed, although the responses did tend to increase slightly so thatthe titers approached the declining antibody levels of the control mice(FIG. 51). At this point, mice were again cured (day 77) with 1, 10 and100 μg 159-dex and subsequently boosted intraperitoneally with 104-BSA,as were the control mice. It is clear that cured mice can withstand thischallenge since although the antibody responses of control mice weresubstantially boosted (as would be expected), those of the cure micewere not significantly changed from pre-challenge levels.

It appears therefore that the effect of 159₁₀-dex₄₀ is not only tosuppress the ongoing anti-159 antibody response but also to inactivatethe specific memory B cells such that they can no longer respond to thechallenge with 104-BSA. This indicates that the suppression occurs atthe level of the specific B cells and is not just an apparentsuppression caused by anti-159 antibodies being absorbed to thecirculating conjugates and thus being effectively removed from the sera.

To test whether there were in fact fewer B cells responding to 159 inthe cured mice than in controls, spleens of sample mice from each groupwere enumerated for antibody secreting cells by the spot ELISA assay.Table 12 indicates that there was indeed a dramatic difference in thenumbers of spleen cells secreting anti-159 antibodies between cured andcontrol animals. In cured animals, the number of spots was negligiblewhereas high numbers occurred in the controls. This indicates that theadministration of 159₁₀dex₄₀ has caused the functional deletion of159-specific B cells which can therefore no longer differentiate intoantibody secreting cells upon subsequent stimulation with specificantigen.

TABLE 12 Ab TITERS COATING STATUS TO 159 ANTIGEN OF MICE OD 405 nm μg/mlSPOTS/WELLS SEM CURE 0.219 100 3 1.5 ″ 50 3.7 0.88 ″ 1 2.3 0.33 ″ 1002.7 0.7 ″ 50 4 1.4 ″ 1 1.3 0.66 ″ 0.231 100 3.3 2.1 ″ 50 3.3 3.3 ″ 1 1.71.7 ″ 100 3.3 1.2 ″ 50 4 1 ″ 1 0 0 CONTROL 2.299 100 31 4.8 ″ 50 21 0.6″ 1 36 5 ″ 100 29 1 ″ 50 25 3.5 ″ 1 ND ND ″ 2.429 100 60 5.8 ″ 50 55 8 ″1 49 1.1 ″ 100 53 2.7 ″ 50 63 4.3 ″ 1 ND ND Spleens from individual micewere enumerated for cells secreting anti-159 antibodies. Two × 10⁵ cellswere added to each well. ND = not determined

Example 12

Treatment of Autoimmune Disease

From the studies described above, the ability of the Immunon technologyto suppress ongoing antibody responses to extrinsic antigens is clear.Its applicability to spontaneous autoimmune processes is established bythe results that follow.

While there are a number of models of autoimmune disease (experimentalautoimmune encephalomyelitis (EAE) for multiple sclerosis, experimentalautoimmune myasthenia gravis, collagen induced arthritis for rheumatoidarthritis, etc.) they all suffer from a common paradigmatic problem: thesymptom complex exhibited in the experimental animal requires theadministration of an extrinsic antigen for the induction of the disease.

In many cases, in order for the symptoms to be maintained, continued,regular administration of antigen is required otherwise the diseaseprocess wanes. The relevance and applicability of these models tospontaneously occurring autoimmune processes in man is unclear. Thereis, however, a model of autoimmune disease in mice (the NZB/NZW mousemodel of human systemic lupus erythematosus—murine lupus) that parallelsthe human with great fidelity. It is spontaneous and does not requirethe administration of extrinsic antigen for its induction ormaintenance; the spectrum of antibodies generated are similar to thatseen in human lupus; the disease manifestations are the same withglomerulonephritis being primary; and, the distribution of disease withregard to the sex of the animal is the same (females develop earlier andmore severe cases of the disease than males). It is this model ofautoimmunity that was chosen to demonstrate the utility of thistechnology with regard to autoimmune disease.

Animals suffering from murine lupus exhibit the production of bothanti-histone as well as anti-DNA antibodies. In preliminary experiments,the distribution of antibodies directed against histone proteins inthese mice was shown to be predominantly limited to the amino terminalregion of H2B. In fact, in greater than 90% of the mice tested theantigenic region of this protein was found to reside between residues 3and 12 inclusively

In order to demonstrate the ability to suppress this response, a peptidereferred to as CI-0084

was synthesized and conjugated to 65,000 molecular weight dextran (fordetails see synthesis section above). This peptide consists of residues2 through 13 of H2B along with two glutamic acids and a cysteine. Theglutamic acids were included in order to render the overall charge onthe peptide neutral at physiologic pH and the cysteine was included forthe conjugation chemistry. The final constructs were then given to micewith already established antibody titers to both histone and DNA.

The protocol for conjugate administration is included in FIG. 52. As canbe seen, in all of the mice that received the suppressive conjugateanti-histone antibody titers were suppressed to background levelswhereas animals that received control conjugates showed no significantchanges (or in many cases actual increases) in their anti-histonelevels. The specificity of suppression is illustrated in FIG. 53 whereinanti-histone responses are shown to be suppressed while anti-DNAantibody levels are essentially unchanged. In addition to thesuppression of circulating antibody titers, enumeration of the B-cellpopulation secreting anti-histone and anti-DNA antibodies in control or“cured” mice was found to parallel the measured antibody levels. Animalstreated with the histone specific suppressive conjugates (which reducedcirculating antibody titers by greater than 95%) were found to have nodetectable cells actively secreting anti-histone antibodies while thecontrol animals had a population of anti-histone antibody secretingcells that were too numerous to quantitate using our standard protocols.Both groups (control and cured) had both equivalent numbers of anti-DNAsecreting cells as well as antibody titers. These data clearlyillustrate the ability of the Immunon technology to suppress aspontaneous, ongoing autoimmune response on an antigen specific basis.

Example 13

Stimulation and Suppression of Fluorescein Specific T-cell Response byFluorescein Substituted Soluble Polymers

Measurements were made of biologically relevant responses of a T-cellline, after exposure to defined, soluble, polymeric molecules containinghaptens capable of binding specifically to the T-cell surface antigenreceptors. The responses obtained in these experiments with T-cell lineswere found to be in close agreement with the expectations based on theImmunon model. This was true for both the dose-response behavior of theT-cells to individual haptenated polymer preparations and thedose-inhibition behavior observed when stimulatory polymers andnon-stimulatory (suppressive) polymers were administrated together. Thefindings confirmed the fact that the general rules of stimulation andcompetitive inhibition implicit in the Immunon theory could be appliedboth to B-cells, which give rise to cells secreting antibody molecules,and to T-cells, which have diverse functions in the regulation of theimmune response.

The T-cells used in these experiments were derived from a human T-cellline (Jurkat), which has been widely used as a model of restingperipheral human T-cells. The Jurkat T-cell was transfected with genesencoding both the alpha and the beta polypeptide chains of afluorescein-specific human T-cell antigen receptor. This transfectedJurkat line was shown to be functional, since it could produce thelymphokine, interleukin-2, upon treatment with conventional T-cellactivators, such as a combination of anti-receptor antibody and phorbolester in the culture medium. When tested for their response to specificantigen, the resulting modified (transfected) Jurkat T-cells were foundto bind soluble fluoresceinated polymers directly to the transfectedantigen receptors on their cell surface. Appropriate solublefluoresceinated polymers, i.e., those of high molecular mass andcontaining a large number of fluorescein epitopes, caused functionalactivation of the T-cell transfectants. Activation of the T-cells bythese soluble polymers was demonstrated by either of two differentassays:

1) Production of T-cell interleukin-2.

2) Production of an intracellular calcium flux.

Soluble polymers of smaller molecular mass and substituted with fewerhapten groups did not activate the transfected T-cells, but werenevertheless potent inhibitors of the activation caused by the larger,more heavily haptenated polymers.

FIG. 54 demonstrates, for a particular pair of fluoresceinated polymers,a representative example of the experimental data described above. FIG.54 (a) shows that a heavily fluorescein-substituted Ficoll preparationof molecular mass over 100 kDa, FL50-Fic150, activated the transfectedJurkat T-cells to produce interleukin-2, as measured by tritiatedthymidine incorporation by an IL-2 sensitive cell line. Thedose-response stimulation curve is bell shaped, as was observed in thesimilar mouse B-cell studies previously described. In contrast, the sameFigure shows that a fluorescein-substituted dextran, FL8-Dex21, of asimilar epitope density but molecular mass well below 100 kDa, was notcapable of stimulating the same transfected T-cells at any comparabledose.

However, FIG. 54(b) shows that when the two polymers were simultaneouslyadded to the transfected T-cells, increasing amounts of thenon-stimulatory smaller polymer can be clearly seen to inhibitincreasingly the activating ability of the larger, stimulatory, polymerin a dose-dependent manner.

Similar activation and inhibitory effects were observed whenintracellular calcium flux was measured for the transfected T-cellsusing soluble fluoresceinated polymers, FIG. 55. In this particularexample, which is representative of a number is similar measurements, alarge highly substituted polymer FL50-Fic150, stimulated the rapidactivation of intracellular calcium flux when added at low or moderatedose (a and b), but not at high dose, (c). A non-stimulatory polymer ofsmaller size but similar epitope density, FL11-Fic46, caused a lack ofresponse by the cells to competitive inhibition (d and e)

The entire contents of all references cited hereinabove are incorporatedherein by references.

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What is claimed is:
 1. A method of making a non-immunogenic constructcomprising at least two copies of an epitope of a T-dependent antigenbound to a pharmaceutically acceptable non-immunogenic carrier, whichcopies bind to a B cell membrane immunoglobulin receptor specific forthe epitope but fail to form an immunon, comprising (a) coupling two ormore copies of said epitope to a nonimmunogenic soluble carrier to yielda conjugate preparation, and (b) removing high molecular weightimmunostimulatory molecules from the conjugate preparation, therebyyielding a construct which is free of high molecular weightimmunostimulatory molecules.
 2. The method of claim 1, wherein theepitope is a peptide epitope.
 3. The method of claim 1, wherein theepitope is a carbohydrate epitope.
 4. The method of claim 1, wherein theepitope is a nucleic acid epitope.
 5. The method of claim 1, wherein theepitope is a glycolipid epitope.
 6. The method of claim 1, wherein thecarrier is dextran, Ficoll, carboxymethylcellulose, poly (D-GLU/D-LYS),albumin, or immunoglobulin.
 7. The method of claim 1, wherein the copiesof the epitope are bound to the carrier by a spacer arm.
 8. The methodof claim 1, wherein the construct comprises from 4 to 30 copies of theepitope.
 9. The method of claim 1, wherein the construct comprises from6 to 14 copies of the epitope.